Detection of Alzheimer's tau protein using localised surface plasmon resonance-based immunochip

Abstract

In this study, we present the detection of tau protein, at room temperature, using a multi-spot localised surface plasmon resonance (LSPR)-based immunochip. To the best of our knowledge, this is the first report of an immunochip for tau protein. The detection method includes fabrication of a gold-capped nanoparticle LSPR chip, formation and functionalisation of a self-assembled monolayer (SAM), immobilisation of a suitable linker, effective blocking of non-specific adsorption, immobilisation of a monoclonal anti-tau antibody (tau-mAb), and finally, the optimum conditions for the immuno-reaction between tau-mAb and the antigen were determined. The method has a high performance, enables detection of tau at 10 pg/mL, lower than the cut-off value of 195 pg/mL (for AD) for tau protein in cerebral spinal fluid (CSF). Further, we demonstrated selectivity of the technique by showing that the introduction of bovine serum albumin (BSA), perhaps the most abundant protein component in serum and CSF, does not interfere with the detection of tau. This method also offers a potential platform for studying tau interactions with other proteins and/or potential drug candidates and could also be easily adapted for detecting phosphorylated tau and other AD biomarkers.

Introduction

Diagnosis of Alzheimer's disease (AD) remains in the hands of medical doctors, who can only at best propose ‘probable Alzheimer's or dementia of the Alzheimer type’ since there is no current test or procedure that is diagnostic. Diagnosis can only be confirmed on autopsy by histopathological features; such as amyloid plaques, neurofibrillary tangles (NFTs), neuronal thread, neuronal and synaptic loss, and brain atrophy in certain regions of the brain [1], [2]. Several biomarkers have been reported [3], [4], [5]. To date, the most extensively studied are amyloid beta (Aβ42), tau and, to a lesser extent, phosphorylated tau in cerebral spinal fluid (CSF) samples, typically using enzyme-linked immunosorbent assay (ELISA) techniques, which are less flexible, costly and labour-intensive [6], [7], [8], [9], [10], [11]. Chip-based technologies have several advantages over the conventional bioanalysis systems because (i) they enable rapid analysis of a large number of samples in a single experiment, (ii) there is reagent economy, and (iii) the signal-to-noise ratio exhibited by micro- or nano-fabricated biochips is much better than that observed for conventional microtiter plate assay systems [12], [13].

Tau is a 50–65 kDa protein. It was first isolated in 1975 as a heat stable protein that promotes assembly, and maintains the structural integrity of microtubules, supports the out-growth of axons and regulates the transport of vesicles and organelles [14], [15]. In the brains of AD individuals, tau proteins become abnormally hyperphosphorylated. It is phosphorylated at more than 20 residues, many (but not all) of which are serine/threonine–proline sites. In healthy individuals, 8–10 of these residues are heterogeneously phosphorylated, and lose the capacity to bind to microtubules. Instead, the phosphorylated tau proteins bind to each other inside nerve cells, tying themselves in “knots” known as NFTs [6], [11]. The first report of CSF tau as a biomarker was published in 1993 [16]. Levels of tau are increased in CSF of AD individuals compared to age-matched controls [6], [7], [8], [9], probably due to neuronal and axonal degeneration or accumulation of NFTs [10]. A cut-off value of 195 pg/mL tau in CSF can accurately differentiate clinically diagnosed AD cases from controls with 89% specificity [8]. Detection of tau, in vitro, is also studied using microscopy, laser light scattering and fluorescence in conjunction with thioflavins (ThS) as the molecular label [17]. Chromatography and SPR have been used in the study of tau–amyloid β interaction [18]. LSPR-based detection techniques are becoming increasingly popular. Several biomolecules have been detected using LSPR immunosensors [19], [20], [21].

In this work, a method based on the immuno-affinity of tau protein towards a monoclonal anti-tau antibody (tau-mAb) immobilised on an LSPR multi-spot nanoparticle chip was employed for the detection of tau protein at room temperature (RT). The approach for LSPR chip fabrication was based on Endo et al.'s [19], [20], with some changes for increased detection sensitivity. Optimisation of each analytical procedure and performance (detection limits, reproducibility, stability of chip and reagents) were examined. Further, the selectivity of the technique by introducing bovine serum albumin (BSA), perhaps the most abundant protein component in serum and CSF [22], was assessed.