In pursuit of degenerative brain disease diagnosis: Dementia biomarkers detected by DNA aptamer-attached portable graphene biosensor

Abstract

Dementia is a brain disease which results in irreversible and progressive loss of cognition and motor activity. Despite global efforts, there is no simple and reliable diagnosis or treatment option. Current diagnosis involves indirect testing of commonly inaccessible biofluids and low-resolution brain imaging. We have developed a portable, wireless readout-based Graphene field-effect transistor (GFET) biosensor platform that can detect viruses, proteins, and small molecules with single-molecule sensitivity and specificity. We report the detection of three important amyloids, namely, Amyloid beta (Aβ), Tau (τ), and α-Synuclein (αS) using DNA aptamer nanoprobes. These amyloids were isolated, purified, and characterized from the autopsied brain tissues of Alzheimer's Disease (AD) and Parkinson's Disease (PD) patients. The limit of detection (LoD) of the sensor is 10 fM, 1-10 pM, 10-100 fM for Aβ, τ, and αS, respectively. Synthetic as well as autopsied brain-derived amyloids showed a statistically significant sensor response with respect to derived thresholds, confirming the ability to define diseased vs. nondiseased states. The detection of each amyloid was specific to their aptamers; Aβ, τ, and αS peptides when tested, respectively, with aptamers nonspecific to them showed statistically insignificant cross-reactivity. Thus, the aptamer-based GFET biosensor has high sensitivity and precision across a range of epidemiologically significant AD and PD variants. This portable diagnostic system would allow at-home and POC testing for neurodegenerative diseases globally.

Keywords: Alzheimer’s disease; aptamer; biosensor; dementia; graphene.

Fig. 1.

Schematic of sensing platform and detection methodology. A schematic of the biosensor testing process utilized throughout the paper. Top-left: Amyloid proteins were immunoprecipitated from homogenized brain tissue from autopsied AD and PD patients. The brain-derived proteins were then applied to the silicone well in the GFET biosensor chip (Top-right). Bottom-left: The 3D models of neurodegenerative amyloid proteins (PDB IDs for Aβ, Tau, and αS are 6cvj, 1xq8, and 2mxu) are shown produced with the software, ChimeraX (–22). Bottom-right: The graphene surface of the GFET chip is functionalized with an aptamer (probe) that binds to the specific analyte (shown as Aβ monomer) and analyte-probe-specific interaction shifts the Dirac point in the plots of the gate voltage vs drain–source currents. The Dirac point shift between the baseline (control I–V curve without an analyte) and the I–V curve in the presence of the biomarker sample is recorded and analyzed by the reader (18).

Fig. 2.

GFET Sensor Characterization via Raman Spectroscopy and AFM. (A) AFM height image of a bare GFET sensor with its section profile (denoted by the white line in A–D). (B) AFM height image of a PBASE functionalized sensor. (C) AFM height image of sensor post fully functionalizing and adding Aβ_1–42. (D) AFM height image of Aβ_1–42 on freshly cleaved mica showing a distribution of lower order oligomers and fibrils. R_q is the RMS roughness of the entire AFM image. All heights in AFM images are between 0 and 30 nm. (E) Raman spectroscopy plot of bare graphene chip on a single 20 μm × 20 μm area. (F) Raman spectroscopy plot of PBASE functionalized graphene on a single 20 μm × 20 μm area.

Fig. 3.

GFET Detection sensitivity and aptamer probe-specificity for Aβ, Tau, and αS proteins. (A–C) The plots A, B, and C show concentration-dependent Dirac shift for Aβ, Tau, and αS, respectively. Each curve is derived from independent experiments of the respective proteins with the same concentrations tested (1 fM, 10 fM, 100 fM, 10 pM, 1 nM, and 100 nM). The dotted black line, the sensing Threshold, indicates an SNR ratio of 3 (3 × the PBS control experiments Dirac shift), 60 mV, 60 mV, and 70 mV for Aβ, Tau, and αS respectively. (D) Plot D is the summary histograms of experimental results supporting the specificity of aptamer probes for their cognate proteins (Aβ_1–42, Tau, and αS). GFET response of synthetic Aβ, Tau, and αS peptides to their specific aptamers (blue: Aβ aptamer, red: Tau aptamer, yellow: αS aptamer are represented in the first three left bars). The next three bars show the significantly lower, nonspecific response for amyloids tested against their nonspecific aptamer. The last bar from the left (gray bar) indicates an average of the results of all three aptamers tested with their nonspecific cognate proteins. The x-axis denotes the analyte protein tested using color-coded aptamers. The positive controls had the correct protein added to the sample (bars to the left of the vertical dotted line). The negative controls are an average of both other proteins added to the incorrect aptamer chip (bars to the right of the vertical dotted line). The result labeled as Avg is an average of each of the negative control nonspecific protein experiments. We illustrate significant P-values between the cross-protein controls and correct protein–aptamer Dirac shift results.

Fig. 4.

Detection threshold for synthetic Aβ, Tau, and αS protein using their specific aptamer probes. (A) Aβ_1–42 at varying concentrations as well as PBS alone (control) were tested using Aβ aptamer functionalized GFET. (B) Tau protein at varying concentrations as well as PBS alone (control) were tested using Tau aptamer. (C) αS protein at varying concentrations as well as PBS alone (control) were tested using αS aptamer. Each plot illustrates the significant P-values between the synthetic protein and PBS buffer control experiments. The Dirac voltage shift threshold line is shown as a reference for a theoretical cutoff signifying positive from negative results (3 × a PBS control results, see Fig. 3). In the case of Aβ, where higher soluble Aβ_1–42 levels correlate more with normal cognition rather than an AD state, the greater Dirac shift will relate to less probable AD diagnosis.

Fig. 5.

Detection threshold and specificity of AD patients autopsied brain-derived Aβ, Tau, and αS proteins. (A) Results of our biosensor against brain-derived Aβ_1–42 proteins isolated from a diagnosed individual with AD at varying concentrations. (B and C) Similar experimental results with brain-derived tau protein and αS protein. (D) A comparison of the different brain-derived proteins against each other. The significance (P-value) of each result is shown with respect to a PBS buffer control. The color of the bars is associated with the aptamer used on the GFET chip (blue Aβ, red tau, yellow αS, gray is an average of all pbs results across aptamers). The solid ΔVg Threshold line is the detection threshold for the Dirac shift in positive samples (3 × a PBS control experiment, see Fig. 3).