Cascade Amplification Biosensor Technology for Detecting MicroRNA Biomarkers of Alzheimer Disease

Duke University, Durham NC

Investigators

Abstract

The goal of this project is to develop a novel molecular diagnostic technology, called “Cascade Amplification by Recycling Trigger Probe” (CARTP), for rapid, multiplex, and ultra-sensitive detection of circulating microRNA (miRNA) biomarkers in clinical plasma samples for early detection of Alzheimer’s disease (AD). Circulating miRNAs have been identified as promising non- or minimally-invasive diagnostic biomarkers in many diseases, including neurodegenerative diseases such as AD. However, because of technical difficulties arising from analytical aspects in the lab, the detection of these small molecules has not been adopted into clinical practice. Common miRNA detection methods, including qRT-PCR, northern blot, microarray, and next-generation sequencing (NGS), are often elaborate, time-consuming, and expensive. There is a need to develop alternative molecular assay strategies that may offer more advantages over conventional methods and have the potential to enhance research in the areas of early detection, screening, and clinical diagnosis. Our laboratory has pioneered the development of a unique homogeneous surface-enhanced Raman scattering (SERS)-based inverse Molecular Sentinel (iMS) nanobiosensor for multiplex detection of miRNAs. The proposed CARTP technique is designed to improve the detection sensitivity of the iMS by amplifying the SERS signal upon detection of targets. Although miRNAs related to AD will be used as the model system, the proposed project will also lead to the development of a generally applicable diagnostic technology for other types of diseases. The following specific aims promote the development of the CARTP technology: (1) Develop the new CARTP molecular analysis method for multiplex detection of miRNA biomarkers; and (2) Technical evaluation of the CARTP for AD diagnosis using clinical samples. In the technique development phase (Aim 1), we will develop and optimize iMS+CARTP nanoprobes for multiplex detection of 3 miRNAs selected from our NGS analysis. The analytical features of merit (specificity, sensitivity, multiplex capability) of the new CARTP technology will be investigated in detail using synthetic targets. In the technical validation phase (Aim 2), the assays will be performed on RNA extracted from clinical plasma samples; results will be compared with NGS data. The performance criteria will include specificity, sensitivity, and multiplex capability provided by the CARTP assay. We will define and test performance measures that could substantiate the expectations of the potential transformative impact of the CARTP technology on AD diagnostics. The proposed technology will lead to a novel “sample-to-answer” analysis approach that is simple and robust to allow successful routine use by minimally trained clinical personnel. The new CARTP technology is capable of enhancing research and translation in the areas of early detection and screening of various diseases beyond AD; it is ultimately better suited for the clinic or point-of-care. The proposed molecular analysis technology represents a significant innovation with transformative potential in biomedical research, diagnostics, and screening.