| Summary: | 碩士 === 國立清華大學 === 化學工程學系 === 103 === This study aims at applying magnetic graphene-based nanocomposite to amyloid-beta (Aβ) biosensor. Aβ is considered as a reliable biomarker for the early diagnosis of Alzheimer’s disease (AD). There is no cure for AD to date, hence, simple and efficient methods for diagnosing AD at its earliest stages is of great importance. Accordingly, a reusable biosensor based on magnetic nitrogen-doped graphene (MNG) modified Au electrode for the detection of Aβ has been developed in this work.
To begin with, nitrogen-doped graphene (NG) was synthesized by heat treatment process from graphene oxide (GO) which was synthesized using modified Hummers’ method. In order to obtain magnetic nitrogen-doped graphene (MNG), magnetic nanoparticles (Fe3O4) were introduced onto NG by co-precipitation. To achieve the best use of functional groups on MNG, ethylenediamine (EDA) was utilized to react with carboxyl groups on MNG to form EDA-MNG by EDC/NHS (1-ethyl-1-(3-(dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide) crosslinking method. Then the antibodies of Aβ were immobilized onto EDA-MNG electrode coating material via SMCC crosslinking method to form antibody-EDA-MNG. In addition, the biosensor was fabricated by coating the antibody-EDA-MNG onto the commercial gold electrode by placing an external magnet at the underside of the electrode for the detection of Aβ, and the biosensor can be easily and conveniently regenerated by switching off the magnetic field used to capture the magnetic materials onto the electrode surface. Finally, the sensitivity, selectivity and repeatability of Aβ biosensor were also investigated in this work.
The first part of this study was the characterization and the investigation of the morphology of EDA-MNG by XRD, XPS, FTIR and SQUID. Firstly, the XRD pattern of EDA-MNG showed obvious peaks of Fe3O4. The six peaks correspond to (220), (311), (400), (422), (511) and (440) planes of crystalline face-centered cubic Fe3O4 nanoparticles. Secondly, the XPS pattern of NG showed the additional peak at 400 eV, which can be attributed to the nitrogen doping from GO. The peaks at 711 eV and 724 eV of MNG and EDA-MNG were attributed to the Fe 2p1/2 and Fe 2p3/2, repectively. Additionally, the EDA-MNG nanocomposites exhibited the superparamagnetic property as well as Fe3O4. Finally, the morphology of EDA-MNG from AFM and TEM indicates the Fe3O4 nanoparticles were immobilized uniformly with a size range from 10 to 20 nm. These charaterizations suggest that the magnetic graphene-based nanocomposites were synthesized successfully.
The second part of this study were the investigations on the EDA-MNG applied in Aβ biosensor using electrochemical method. On one hand, the electrochemical potential window of the biosensor between -0.2 to 0.6 V was obtained by cyclic voltammetry (CV). On the other hand, differential pulse voltammetry (DPV), an electrochemical technique which can reduce the background noise was utilized to detect the Aβ concentration. In terms of sensitivity, the calibration curce was linear within the range from 5 pg mL-1 to 800 pg mL-1 with R2 = 0.9977, covering cut-off level of Aβ and a detection limit of 5 pg mL-1 had been achieved. In terms of selectivity, the influence of detection in the presence of 30 μM asctrbic acid (AA) and 30 μM uric acid (UA) can be neglected. In terms of reusability, the RSD of the current response of reconstruction was 1.40% (n=50) and the RSD of the current reponse of Aβ deteion was 2.80% (n=6), indicating that the biosensor can be reused with good reproducibility and stability.
In summary, an Aβ biosensor with high sensitivity, high selectivity and repeatability was fabricated successfully. The fabricated biosensor for Aβ detection not only improves the detection performance but also reduces the cost and shortens the response time. Furthermore, this biosensor could be utilized for early diagnosis of Alzheimer’s disease because the detection range of this biosensor covers the Aβ levels of normal people and patients.
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