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Title: 開發胺基改性氧化石墨烯之電漿子胜肽適體感測器於臨床血清樣本中人類絨毛膜促性腺激素檢測
Development of amine modified graphene oxide based peptide SPR aptasensor for human chorionic gonadotropin in clinical serum detection
Authors: 邱南福
Chiu, Nan-Fu
Kuo, Chia-Tzu
Keywords: 胺基修飾氧化石墨烯
Amine modified graphene oxide
Surface plasmon resonance biosensor
human Chorionic Gonadotropin
Issue Date: 2017
Abstract: 本論文提出一個簡易的胺基修飾法之氧化石墨烯(amine modified graphene oxide, GO-NH2),作為非免疫反應、超高靈敏度表面電漿子共振生物感測器之感測層,用以偵測人類絨毛膜促性腺激素(hCG),此激素已用在多種孕婦問題,諸如:早產、唐氏症、子宮外孕,也和卵巢癌、乳癌、睪丸癌等性腺相關癌症有關聯,此激素在健康未懷孕個體的血漿中約含有0.45 nM 的濃度。而本實驗利用胜肽取代抗體,擁有能夠保存於相對高溫的空間以及製造成本便宜等優點,為了達到早期檢測以及利於商業化, GO-NH2修飾的生物感測器是同時具有高親和性與高靈敏度優勢的技術。 修飾的步驟將GO中的羥基利用亞硫醯氯(SOCl2)的氯元素取代,生成中間產物氯基修飾氧化石墨烯(GO-Cl),再利用氨水將氯基取代為胺基,採用X射線光電子能普儀(X-ray photoelectron spectroscopy, XPS)驗證,其結果氮元素比例增加至5.7%,而氧元素的比例由原本約27%降至約17%。傅立葉轉換紅外光譜(Fourier transform infrared spectroscopy, FTIR)分析,在1064 〖cm〗^(-1)、1579 〖cm〗^(-1)以及3361 〖cm〗^(-1)的波段均得到氨基的震動吸收峰。藉由共振角模擬的折射值結果為1.55 + C_1 λ/n i,而電化學阻抗模擬得到R_(GO-NH_2 )的阻抗為89.16 Ω,相較於R_GO的733.6 Ω降低了8.23倍。 重組蛋白實驗中,GO-NH2對於本實驗使用的胜肽親和性比起之前提出的羧基修飾氧化石墨烯(GO-COOH)高出2.45倍,比起傳統晶片,GO- NH2在2 nM的濃度下反應提高了約2.68倍,而線性迴歸斜率提高了1.5倍,並在臨床血清檢測則發現唐氏症樣本的反應高於非唐氏症樣本約1.6倍,並且都擁有良好的迴歸係數。希望在未來可以規劃成唐氏症早期篩檢系統協助臨床醫師進行診斷。
In this work we present a facile processes amine modified graphene oxide (GO-NH2) as an ultra-sensitive surface plasmon resonance (SPR) probe grafting layer for non-immunization sensing. The GO-NH2 based SPR biosensor was applied in detection of human Chorionic Gonadotropin (hCG), relevant to pregnant disease. Such as premature birth, ectopic pregnancy, and Down's syndrome. It’s also relevant to some cancer about gonad, like ovarian cancer, breast cancer, or testicular cancer. The concentration of hCG in blood plasma of healthy and non-pregnant is around 0.45 nM. Replacing antibody by non-immunization peptide got an opportunity of preserve the probe in relative higher temperature and much lower cost than antibodies. In order to diagnose the patient in the early state and benefit for commercial situation, the characteristic of high resolution GO-NH2 modified bio-chip is a novel and advantage technique. The first part of two-step modified approach is substitute the hydroxyl with chloride on GO. The chloride functional graphene oxide (GO-Cl) is obtained through the reaction of GO and thionyl chloride (SOCl2). After that, we utilize ammonia water as nitrogen precursor, and the chloride groups are replaced by amino groups. The primary of facial amine group is identified by X-ray photoelectron spectroscopy (XPS). The ratio of nitrogen and carbon in our experimental parameter is increase to 5.7%, and the ratio of oxygen is changed from 27% to 17%. In the Fourier transform infrared spectroscopy (FTIR) experiment. The absorbed peak of 1064 〖cm〗^(-1), 1579 〖cm〗^(-1), and 3361 〖cm〗^(-1) that absorbed by N are found. The simulation of complex refractive index and the impedance of GO-NH2 is 1.55 + C_1 λ/n i and 89.16 Ω. The recombinant protein experiment shows that GO-NH2 sensor-chip is 2.45 times about the affinity of peptide. Comparing in non-immunization diagnostic, the response at 2 nM is 2.68 times greater, and the slope of GO-NH2 linear regression is 1.5 times higher than commercial chip. Last, the clinical blood serum experiment shows high linear regression coefficients.
Other Identifiers: G060448026S
Appears in Collections:學位論文

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