Division of Research, College of Osteopathic Medicine USA
Title: Nanodiagnostic Approach to Detection of Plasma Biomarkers: Potential Screening tool for Early Diagnosis of Alzheimerâ€™s Disease
Esther Lim, MD, MBA is currently an associate professor of radiology at PCOM and works in collaboration with Dr Hui Mao, PhD, Professor of Radiology and Biomedical Engineering and the Director for the Molecular Imaging, Biomarkers, and Probe Development at Emory University. Dr Yuanchen Li, PhD is a staff scientist at Emory University School of Medicine with expertise in anti-biofouling polymer synthesis for nanomaterial coating and biomarker targeted imaging, drug delivery and therapy.
Alzheimer’s Disease (AD) imposes a huge economic burden with its diagnostic and therapeutic challenges. The currently available diagnostic tools are expensive, invasive, and insufficient for early screening. At present, levels of amyloid-β (Aβ) in the brain can only be assessed reliably via Positron Emission Tomography (PET) imaging or by measuring amyloid-β (Aβ) levels in the cerebrospinal fluid. However, such invasive and expensive approach is impractical for patients with no obvious symptoms. Recent literature demonstrating the potential clinical utility of plasma biomarkers in predicting brain amyloid-β (Aβ) burden has drawn increasing interest among nanoparticle researchers. Development of minimally invasive and ultrasensitive detection system for early diagnosis of AD is critical for formulating optimal therapeutic strategies early in the progression of the disease. Using anti-biofouling magnetic nanomaterials, we have constructed a novel, simple and efficient method that could serve as a screening tool.
The anti-biofouling polymer coated Iron Oxide Nanoparticles and their targeting antibody conjugates were prepared as described in the literature. The Fluorescein Isothiocyanate (FITC) labeled amyloid-beta peptide 1-40 [Aβ(1–40)] and Tetramethylrhodamine-5-(and6)-isothiocyanate (TRITC) labeled peptide 1-42 [Aβ(1–42)] were dissolved in Phosphate Buffered Saline (PBS) with Fetal Bovine Serum (FBS) at 50mg/mL to mimic the human serum environment. They were then incubated with antibody (Ab)-conjugated IONPs (at final iron concentration of 0.2 mg/mL) or antibody- conjugated Dynabeads for 3 hours before magnetic separation of particles. The separation efficiency (SE) was calculated as the weight ratio of captured peptide to spiked. The protein quantification was verified using micro bicinchoninic acid (BCA) protein assay kit. Furthermore, insulin was purposely added to PBS with FBS as interference to demonstrate the capture specificity of Ab-conjugated IONPs.
The SE of anti-biofouling IONP for Aβ(1–40) in PBS with FBS at 0.1, 0.2, 0.5, 1, 2, 5, and 10 microgram/mL were.in the rage of 86-97% using fluorescence signal and with microBCA protein assay kit. When insulin was added to Aβ(1–40) mixture, the separation efficiency of insulin for IONP was only 4.5, 4.7, 6.2, 6.5, 7.7, 9.8, and 12.3% while dynabeads showed no difference in the separation efficiency for both Aβ(1–40) and insulin. Similar results were obtained for Aβ(1–42) and IONP with minimal isolation of insulin. Dynabeads again showed indiscriminate separation of Aβ(1–42) and insulin. The sensitivity for our antibody conjugated IONP detection system is calculated at 88-98.5% and false positive rate of 7.5-11.2%. The commercial dynabeads, however, showed sensitivity of only 27-39% and false positive rate of 48.3-55.4%.
We have developed a highly sensitive and specific Iron Oxide Nanoparticle (IONP) based system with potential application for early AD diagnosis in human serum. Its ability to selectively detect AD markers at minute concentration and with additional benefit of low cost and scalability, it can serve as a non-invasive tool for population-wide screening for AD.