The sensitivity was lower, as the NH3of the CLU will donate electron charge at the interface with SPCE electrode. and a linear concentration range of 1100 pg/mL with good sensitivity. Detection of CLU in spiked human plasma was demonstrated with satisfactory recovery percentages to that of the calibration data. The proposed method facilitates the cost-effective and viable production of label-free point-of-care devices for the clinical diagnosis of AD. Keywords:clusterin, electrochemical biosensor, label-free immunosensor, cyclic voltammetry, square wave voltammetry, screen-printed electrodes, Alzheimers disease == 1. Introduction == Clusterin (CLU), also known as Apolipoprotein J (Apo J, a highly conserved heterodimeric glycoprotein with molecular weight Rabbit Polyclonal to PEBP1 of 7580 kDa), has attracted significant scientific attention as it is involved in lipid export during cell differentiation and cell death [1] and can stabilize stressed proteins in a refolding-competent state [2]. It is also implicated in aging and age-related diseases such as neurodegeneration, diabetes, and atherosclerosis [3,4,5], and acts as a biomarker of cellular senescence and oxidative stress [6]. CLU content in the plasma is found to be associated with atrophy of the entorhinal cortex, baseline disease severity, and rapid clinical progression in Alzheimers disease (AD) [7]. It is also found in longitudinal brain atrophy in mild cognitive impairment [8]. In addition, CLU gene variants in association with amyloidogenic peptides and CLU contributes to limit amyloid- (A) species misfolding and facilitates their A 740003 clearance from the extracellular space [9]. To date, most biomarker studies on A 740003 AD have focused on the use of cerebrospinal fluid (CSF) and blood plasma levels of -amyloid, tau, and phosphorylated tau in relation to clinical symptoms and survival as diagnostic specimens [10,11,12,13,14,15]. However, prominent companies and research groups A 740003 are now realizing that the simplistic amyloid cascade hypothesis that was reasonable to propose 26 years ago is not a reasonable hypothesis any longer [16,17,18]. Besides, AD biomarker tests are either highly invasive (requiring CSF collection) or expensive and labour-intensive (neuroimaging), making them unsuitable for use in primary care, clinical office-based settings, or to assess drug efficacy in clinical trials [19]. Note that the current clinical diagnosis for AD blood test is used to rule out other conditions that may be responsible for mental impairment symptoms in suspected patients, such as thyroid hormones and vitamin B12 levels, not the direct test of AD biomarkers mentioned above. Hence, the development of a rapid and cost-effective means of providing routine screening of elderly patients for AD is of paramount importance. An initial blood-based screening method for AD will be able to reduce the financial burden of AD diagnosis in coming decades by a substantial drop of the need for follow-up referral to expensive neuroimaging or CSF analysis [20,21]. Of the various plasma biomarkers investigated for AD, clusterin has been one that holds promise. CLU is the third most associated late-onset Alzheimers disease (LOAD) risk gene according to the AlzGene database [22]. In AD, various studies have shown elevated CLU levels in brain and CSF. Thambisetty et al. (2010) found that CLU is associated with both hippocampal atrophy in AD and mild cognitive impairment (MCI) subjects along with fast progressing, or more aggressive, AD. Evidence from human cerebrospinal fluid, post-mortem brain, and transgenic animal models suggests a plausible link between CLU and AD pathology [7]. To date, the detection of clusterin has been performed by radioimmunoassay, immunohistochemistry, in situ hybridization, Western blotting, and cDNA microarray (Table 1) [23,24,25,26,27,28]. Chung et al. has shown a proof-of-principle, semi-automated, microfluidic point-of-care method of KIM-1 and Cystatin C detection in urine, and proposed that CLU can also be adapted in their method [29]. However, no report has been found for the electrochemical detection of clusterin. == Table 1. == Detection of clusterin (CLU) by various methods. IHC: immunohistochemistry; ISH: in situ hybridization; WB: Western blotting; cDNA-MA: cDNA microarray; NB: Northern blotting; LN: lymphoid neoplasms; ALCL: anaplastic large-cell lymphoma. Herein, we report for the first time an electrochemical immunosensor for the detection of CLU. A simple, low-cost biosensor was developed by modifying the carbon electrode with 1-pyrenebutyric acidN-hydroxysuccinimide ester (Pyr-NHS) and immobilizing F(ab)2fragments of CLU antibody to detect CLU antigen specifically. The Pyr-NHS linker utilizes a bifunctional molecule containing succinylimide ester and a pyrene moiety to bind proteins to the carbon surface. Pyrene attaches to the carbon surface by means of the – stacking or hydrophobic interactions and does not significantly disturb the electronic structure of the screen-printed carbon working electrode. F(ab)2fragments of an antibody possess some advantages over whole antibody in terms of close proximity to the surface (within Debye length limit) [30], higher structural stability.