A New Optical Interferometric Biosensing System Enhanced with Nanoparticles for Alzheimer's Disease in Serum

Abstract

In this scientific work, we demonstrate, for the first time, a new biosensing system and procedure to measure specifically the total Tau (T-Tau) protein in serum, one of the most relevant biomarkers of Alzheimer's disease (AD). AD is a progressive brain disorder that produces neuronal and cognitive dysfunction and affects a high percentage of people worldwide. For this reason, diagnosing AD at the earliest possible stage involves improving diagnostic systems. We report on the use of interferometric bio-transducers integrated with 65 microwells forming diagnostic KITs read-out by using the Interferometric Optical Detection Method (IODM). Moreover, biofunctionalized silicon dioxide (SiO₂) nanoparticles (NPs) acting as interferometric enhancers of the bio-transducers signal allow for the improvement of both the optical read-out signal and its ability to work with less-invasive biological samples such as serum instead of cerebrospinal fluid (CSF). As a result, in this paper, we describe for the first time a relevant diagnostic alternative to detect Tau protein at demanding concentrations of 10 pg/mL or even better, opening the opportunity to be used for detecting other relevant AD-related biomarkers in serum, such as β-amyloid and phosphorylated Tau (P-Tau), neurofilaments, among others that can be considered relevant for AD.

Keywords: Alzheimer’s disease; SiO2 nanoparticles; interferometry; optical biosensor; serum Tau protein; silanization.

Figure 1

Biofunctionalization of SiO₂ NPs with αTau antibodies. (A) Depiction of the silanization process of SiO₂ NPs until the carboxyl functional group is obtained. (B) Representation of the final steps in the biofunctionalization process of the NPs where the G-protein and αTau antibodies are attached. Abbreviations: 1-Ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), 2-(N-morpholino) ethane sulfonic acid buffer (MES) and PBS.

Figure 2

Outline of the protocol followed for the competitive assay. (A) Picture of 65 200-micron SU8 “BICELLs” diagnostic KIT used for the assays. (B) Process of protein immobilization on the sensing area of BICELLs by oxygen plasma activation. (C) Diagram of sample preparation in PBS and serum. (D) Depiction of the last steps of the competitive assay and the graph representing the inversely proportional relationship between the ΔIROP (%) signal and Tau concentration. Abbreviations: Biophotonic Sensing Cell (BICELL), phosphate buffer saline (PBS), and Increased Relative Optical Power (IROP).

Figure 3

Size distribution of SiO₂ NPs biofunctionalization process. Depiction of the increase in NPs size in the different steps of the biofunctionalization protocol. Abbreviations: Nanoparticles (NPs).

Figure 4

Specificity antibodies assays. The image above depicts the signal of the αTau antibody on the different proteins tested (Tau, BSA, and p24). The middle image indicates the signals of the αBSA antibody on these proteins, and the lower image corresponds to the signal of the αp24 antibody. Abbreviations: Increased Relative Optical Power (IROP) and Bovine Serum Albumin (BSA).

Figure 5

αTau nanoparticles calibration curve. Representation of the signal of the nanoparticles at different concentrations (1 × 10⁴ to 1 × 10⁷ NPs/µL) against immobilized Tau diagnostic KITs.

Figure 6

Specificity anti-Tau NPs assays. Recognition of αTau NPs over Tau and BSA at one and two hours of incubation. Abbreviations: Increased Relative Optical Power (IROP) and Bovine Serum Albumin (BSA).

Figure 7

Competitive assays in PBS and serum. (A) Linear fitting between Tau concentration and ΔIROP (%) in PBS, with a 95% confidence interval and a 95% confidence band. (B) Linear fitting between Tau concentration and ΔIROP (%) in serum, with a 95% confidence interval and a 95% confidence band. The background signal level is plotted on the graph. Abbreviations: Increased Relative Optical Power (IROP) and Phosphate Buffer Saline (PBS).

Figure 8

SEM images of competitive assays in PBS. (A) BICELL SEM image with a magnitude of 2K for a high concentration of Tau. (B) SEM image of the same BICELL with a magnitude of 10K. (C) BICELL SEM image with a magnitude of 2K for a medium concentration of Tau. (D) SEM image of the same BICELL with a magnitude of 10K. (E) BICELL SEM image with a magnitude of 2K for a low concentration of Tau. (F) SEM image of the same BICELL with a magnitude of 10K.