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Alzheimer's disease (AD) is the most common neurodegenerative disease among the elderly. Abnormal aggregates of both β-amyloid peptide (Aβ) subtypes, Aβ 42 and Aβ 40 , are the typical neuropathology hallmarks of AD. However, because of the lack of specific recognition elements such as an antibody and aptamer, it is difficult to differentiate and determine the oligomers of Aβ 42 and Aβ 40 in clinic. In this paper, we developed a planar bilayer lipid membrane (BLM)-based electrochemical biosensor. According to the dynamic differences on oligomer-induced BLM damage, both low-mass, soluble oligomers of Aβ 42 and Aβ 40 (L-Aβ 42 O and L-Aβ 40 O) were measured in turn by electrochemical impedance spectroscopy. The BLM was supported by a porous 11-mercaptoundecanoic acid layer on a gold electrode, which amplified the impedance signal corresponding to the membrane damage and improved the detection sensitivity. The weakly charged surface of the BLM ensured the low non-specific adsorption of coexisting proteins in cerebrospinal fluid (CSF). Using the electrochemical biosensor, L-Aβ 42 O was determined within 20 min, with a linear range from 5 to 500 pM and a detection limit of 3 pM. Meanwhile, L-Aβ 40 O was determined within 60 min, with a linear range from 60 pM to 6.0 nM and a detection limit of 26 pM. The recoveries in oligomer-spiked artificial CSF and human CSF samples confirmed the accuracy and applicability of this proposed method in clinic. This work provides an antibody-free, highly selective, and sensitive method for simultaneous detections of L-Aβ 42 O and L-Aβ 40 O in real CSF samples, which is significant for the early diagnosis and prognosis of AD.

Phosphorylated proteins play important roles in the pathogenesis of Alzheimer's disease (AD). The most abundant constituent in AD's brain deposit is the amyloid-β 40 peptide (Aβ 40 ). Based on it, the degree of phosphorylated Aβ 40 in body fluids (e.g., cerebrospinal fluid, CSF), which is defined by the ratio of phosphorylated Aβ 40 to total Aβ 40 (pAβ 40 /tAβ 40 ), is anticipated to be an index for early diagnosis of AD. The major challenge in pAβ 40 /tAβ 40 detection is the large concentration difference between two Aβ 40 forms in the real samples, which usually requires multichannel equipment and complicated detection process. In this paper, we revealed the unexpected close affinities of the anti-Aβ 40 antibody to Aβ 40 (40.2 nM -1 ) and to pAβ 40 (42.3 nM -1 ). Based on it, a convenient coimmunocapture and electrochemical quantitation of tAβ 40 and pAβ 40 was achieved on an anti-Aβ 40 antibody immobilized Au electrode (anti-Aβ 40 /Au). Once Aβ 40 and pAβ 40 were synchronously captured on the anti-Aβ 40 /Au electrode, the tAβ 40 levels in CSF samples were quantified with electrochemical impedance spectroscopy. With the signal amplification from Cd 2+ /Ti 4+ -functionalized titanium phosphate nanospheres (Cd 2+ /Ti 4+ @TiP) which was selective conjugated to pAβ 40 , concentrations of low abundant pAβ 40 as low as 1 fM were readily measured by square wave voltammetry. Our results reveal that despite the concentrations of tAβ 40 and pAβ 40 fluctuate in each individual case, the concentration ratios of pAβ 40 /tAβ 40 in CSF samples from AD patients are significant larger than those from healthy donors. It demonstrates that the degree of phosphorylated Aβ 40 is hopeful to be an effective index for evaluating the AD progress and improving the accuracy of clinic AD diagnosis.