Gelsolin bound β-amyloid peptides_{(1–40/1–42)}: Electrochemical evaluation of levels of soluble peptide associated with Alzheimer's disease

Highlights

• A sensitive electrochemical method for the detection of Aβ_{1–40/1–42} was presented.

• Gelsolin was employed for the specific recognition of these peptides.

• A HRP–Au–gelsolin bioconjugate was prepared as the detection nanoprobe.

• This methodology realized the assessment of Aβ_{1–40/1–42} levels in AD rat brains.

Abstract

A method for the highly sensitive determination of soluable β-amyloid peptides (Aβ_{1–40/1–42}) that employs a detection bioconjugate of HRP–Au–gelsolin as the electrochemical nanoprobe is presented. Contrary to previous detection notions that utilized antibodies, which could specifically recognize the N- or C-terminus of peptides, we demonstrate herein that the reported specific binding between gelsolin and Aβ might provide an alternative way to evaluate the peptides sensitively and selectively. The HRP–Au–gelsolin nanohybrid was designed by one-pot functionalization of Au nanaoparticles (NPs) with horseradish peroxidase (HRP) and gelsolin. Through a sandwich-type sensor array, soluble Aβ_{1–40/1–42} were captured onto the array due to the interactions between targeted peptides and surface-confined gelsolin and electrochemical signals were amplified by abundant attachments of HRP labeled on AuNPs, which could specifically catalyse its substrate, 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H₂O₂ to give rise to measurable signals. The proposed gelsolin-bound Aβ methodology displayed satisfactory sensitivity and wide linear range towards Aβ_{1–40/1–42} with a detection limit down to 28 pM, which are verified to be sensitive-enough for the assessment of Aβ levels both in normal and Alzheimer's disease (AD) rat brains. Experimental results indicated that compared with normal group, soluble β-amyloid peptide levels in cerebrospinal fluid (CSF) and targeted brain tissues of AD rats all declined with differentiable degrees. In short, the newly unfolding strategy presents valuable information related to pathological events in brain and will exhibit a braw perspective for the early diagnosis of AD process.

Introduction

Alzheimer's disease (AD), an age-related neurodegenerative condition, was associated with an early impairment in memory and was the major cause of dementia in the elderly (Jiang et al., 2013). As the most common neurodegenerative disease, it has been estimated an increase of up to 106.8 million people by 2050 (Yang et al., 2013). The main characteristic features of AD include gradual loss of cognitive function and synaptic integrity, selective neuronal death and abnormal formation of neurotic and core plaques in the cerebral cortex (Hardy and Selkoe, 2002, Liu et al., 2014). However, so far, no effective treatment for this neurodegenerative disorder has been found and developed.

The pathological features of AD include extracellular neuritic plaques containing β-amyloid peptide (Aβ) and intracellular neurofibrillary tangles (NFTs), which are composed of hyperphosphorylated microtubule-associated protein tau. Experimental data from both in vitro and in vivo studies have indicated that aggregated Aβ initiate a pathogenic cascade that ultimately leads to neuronal loss and dementia (Glenner and Wong, 1984). Amyloid associated with Alzheimer's disease consists of thin fibrils of polymerized Aβ with an ordered β-sheet pattern in AD brains (Ranjini et al., 2012). The actual form of Aβ that causes the damage is most likely a small dimeric or higher oligomeric species with internal β-sheet structure (Selkoe, 1996). Aβ variants, such as Aβ_1–40 and Aβ_1–42, are produced by sequential cleavage of APP by β- and γ-secretases. Inhibitors of β- or γ-secretase or modulators of γ-secretase result in the lowering of central Aβ_1–40 and Aβ_1–42 levels and are therefore pursued as potential disease modifying treatments for AD (Citron, 2010, Niva et al., 2013). Therefore, the two Aβ variants are nowadays widely believed to be important biomarkers and drug targets for AD research and therapy.

Under such circumstance, with the development of amyloid-sensitive ligands, new approaches aimed at assessing amyloid plaque aggregation are thus developed to lessen the burdens of both society and family, including methods that indirectly estimate levels of brain amyloid plaques from Aβ levels in plasma or cerebral spinal fluid (CSF) (Rodrigue et al., 2009). However, most of these methods belong to postmortem identifications, which are often inevitably influenced by the age of the cohort sampled and the method of defining disease pathology (Bennett et al., 2006, Thal et al., 2006). Currently, biochemical assays such as polyacrylamide gel electrophoresis (PAGE), immunoprecipitation, enzyme-linked immunosorbent assay (ELISA) and also non-biochemical techniques, incluindg surface plasmon resonance (SPR), mass spectrometry, capillary electrophoresis have been employed to detect Aβ species from body fluids and cell media (Xia et al., 2010, Golde et al., 2000, Munishkina and Fink, 2007, Picou et al., 2010). Although high sensitivity, selectivity and reliability of ELISA seems more attractive, inherent shortcomings of this technique such as relatively expensive enzyme-linked antibody for Aβ recognition and carcinogenic substrate for chemiluminescent detection should not be ignored. Alternatively, electrochemical biosensors have been considered as a promising technique because of their simplicity, rapid response, and potential ability for real-time and on-site analysis (Zhang et al., 2008), which are typically labeld with an electroactive species to generate corresponding electrochemial signals. Hence, labels that are designed for signal amplification have always been a favorable candidate in the biosensor fabrication, including functionalized liposomes, enzymes and various nanomaterials (Yu et al., 2006, Lu et al., 2008). For example, Liu and co-workers presented a sensitive and selective electrochemical immunosensor for the detection of both Aβ_1–42 and total Aβ using p-AP redox cycling by tris (2-carboxyethyl) phosphine (Liu et al., 2014), which achieved a low detection limit of 5 pM for Aβ_1–42 and this value was comparable or even lower than other non-electrochemical methods.

Discovery of strong interactions between two completely different proteins led to suggestions that the specific affinity could be employed in a similar way as antibody–antigen interaction and hence develop a novel methodology for the detection of one of these proteins. Compared with other detection strategies, this notion emblems no complicated substances or chemical bonds that would be invovled to capture Aβ and therefore becomes much easier to perform. In 1999, Chauhan's group has found that a secretory protein named gelsolin, can bind to soluable Aβ peptide in a concentration-dependent manner (Chauhan et al., 1999). It was found that more gelsolin was taken, more Aβ would be bound until the binding reached the saturation and more significantly, this specificity was proved to be applicable for both Aβ_1–40 and Aβ_1–42 monomers, not their oligomeric and fibril forms. Since Aβ_1–40 and Aβ_1–42 possess more significance toward AD occurance and development in human, this specific binding between Aβ_{1–40/1–42} and gelsolin, somewhat like the interaction between antibody and antigen, would be applicable to fabricate a novel electrochemical immunoassay for the detection of total amount of Aβ_1–40 and Aβ_1–42.

Herein, on the basis of our previous work, we reported the design and evaluation of an alternative approach for electrochemical protein assay employing platforms featuring well-dispersed Au nanoparticles (NPs). The specific recognition of our target, Aβ_{1–40/1–42} was achieved using gelsolin that can specifically bind to Aβ_{1–40/1–42}, as the recognition element for further insight into the floating of Aβ levels in the process of AD. Accordingly, a nanohybrid of HRP–Au–gelsolin was prepared by one-pot in situ functionalization of AuNPs with HRP and gelsolin and used as the detection probe to capture Aβ_{1–40/1–42}. Signal amplification was achieved via enormous loading of HRP on the AuNPs and its strong catalytic activity towards its substrate TMB to produce measurable redox currents. The results indicated that the established gelsolin-based electrochemical assay possesses resonable sensitivity and selectivity, which has met the requirement of evaluating dynamic changes of Aβ_{1–40/1–42} levels in CSF, brain tissues of both normal and AD rats.