In this study we attempted

In this study, we attempted to develop a simple, rapid, label-free, quantitative, and cost-effective method for the detection of Aβ. As pointed out in the literature [19,27], use of electrochemical biosensors is one of the most realistic candidates for achieving this end. In the course of our study on the interaction between Cu2+ and Aβ, we discovered a 19-residue peptide that can accelerate the fibrillization of Aβ, which we call AFPP (amyloid-β fibrillization promoting peptide). We have now prepared N-cysteinyl AFPP (AFPPcys) and immobilized it on a Au electrode in an attempt to accumulate Aβ(1–40) on it and to quantify the deposited Aβ(1–40). After incubation of Aβ(1–40) with the AFPPcys-modified electrode for half an hour, we added Cu2+ to the solution and allowed it to bind. We then washed away the free Cu2+ and carried out cyclic voltammetry (CV) measurements. The results demonstrated that the peak current and peak area are proportional to the concentration of Aβ(1–40) in the range of 0.1–5μM. When amylin or islet amyloid polypeptide, another amyloid fibril forming polypeptide that can bind to Cu2+[22], was used as the target instead of Aβ(1–40), no voltammetric response was observed. In addition, the AFPPcys-modified Au electrode could be reinitialized simply by washing out the deposited Aβ(1–40) and Cu2+ with NaOH and EDTA solutions. The reused electrode demonstrated good reproducibility. These results indicate that this promising electrochemical method provides simple, rapid, selective, and cost-effective quantification of Aβ, although the current sensitivity does not reach the concentration of Aβ found in bodily fluids such as CSF and plasma.
Materials and methods
Results and discussion
Conclusions This work demonstrated the electrochemical detection of Aβ(1–40) at concentrations down to 0.1μM (5×102ng/mL) using an AFPPcys-modified electrode and Cu2+. Washing with NaOH and EDTA solutions easily reinitializes the AFPPcys-modified electrode, which gives good, reproducible results. This method provides rapid, simple, label-free, quantitative, and cost-effective detection of Aβ(1–40). Although Aβ(1–40) was chosen to establish the new assay method, our methodology is easily applicable to other Aβ CGP-41251 of different length, such as Aβ(1–42) and Aβ(1–28). Unfortunately, the sensitivity of this method is insufficient at present for use with levels of Aβ(1–40) found in bodily fluids, such as CSF and plasma. However, there is still room for improvement in the electrode and sampling system. We believe that our method will be valuable for the low-cost early diagnosis of AD when its sensitivity is improved.
Conflict of interest
Acknowledgements This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers 23500571 and 26350552 to S.F. The authors would like to thank Enago (www.enago.jp) for the English language review.
Introduction Biosensors as a diverse collection of bioanalytical devices for detecting and quantifying biomolecules, have been widely used in many areas, such as clinical and home-based diagnoses and environmental monitoring [1–3]. Typically, a biosensor consists of three main parts: a bio-recognition component, a signal transducing component and an output system. Among them, the bio-recognition component is responsible for the identification of the presence and quantification of analytes via specific interactions with analytes by using specific biological elements like proteins, nucleic acids and tissues [4,5]. Thus effective immobilization of these biological elements onto solid surfaces is an important step in biosensor fabrication. Considerable efforts have been devoted to endowing the supporting material surfaces with bio-recognition ability by attaching biomolecules covalently [6,7]. Nowadays, novel paper-based biosensors have emerged with potential as easy-to-use, rapid and inexpensive point-of-care devices [8,9]. Compared with commonly used substrate materials for biosensors, its advantages include low production cost, intrinsic water wicking ability and feasibility of patterning by printing technology [10,11]. So far, in paper-based sensor and paper-based ELISA plate design and fabrication the most used techniques for immobilizing biomolecules are based on physical adsorption. However, physical adsorption of biomolecules has an unavoidable weakness that it could not always promise reproducible results because biomolecules are weakly bound to paper fibers and could be easily washed off [12]. In order to significantly improve the performance of paper-based devices for quantitative bioanalysis, effective and chemically reliable methods for immobilizing a broad range of biomolecules on paper sensors, mostly immobilization via covalent bonding, need to be explored.