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標題: | 利用光電傳感生物感測器於高鹽溶液克服德拜屏障效 應測量生物指標分子 An Optoelectronic Biosensor for Detecting Biomarkers in High Salt Buffers beyond Debye Hückel Screening Effect |
作者: | Yen-Hua Lee 李彥華 |
指導教授: | 陳逸聰(Yit-Tsong Chen) |
關鍵字: | 場效電晶體,生物感測器,光電傳感,二硫化錫,適體,上轉換奈米 粒子,德拜屏蔽效應, field-effect transistor,biosensor,optoelectronic,tin disulfide,aptamer,upconverting nanoparticle,Debye-Hückel screening effect, |
出版年 : | 2020 |
學位: | 碩士 |
摘要: | 場效電晶體(field-effect transistor, FET)秉持其高靈敏度及即時測量(real-time)等優點,於生物感測器(biosensor)領域已有廣泛的利用。其利用目標分子所具備之電荷所產生之電場,誘使電晶體元件內之電流載子(charge carrier)於電流通道內產生變化,進而取得生物訊號。然而FET-biosensor於高鹽環境中,溶液內部帶有之離子會屏蔽目標分子產生之誘導電場,導致電晶體無法測得訊號,此即德拜許可屏蔽效應(Debye-Hückel screening effect)。於高鹽環境中,若目標分子之所持電荷位於元件表面之德拜長度(Debye length, λD) 外,電荷將被大幅屏蔽。為了解決FET-biosensor於高鹽環境中無法測量的問題,我們設計一全新之測量機制──上轉換奈米粒子之光電傳感生物感測器(upconverting nanoparticle based optoelectronic biosensor),此感測器元件包括三大部分:二硫化錫(tin disulfide, SnS2)場效電晶體、DNA適體(aptamer)及上轉換奈米粒子(upconverting nanoparticle, UCNP),利用修飾於元件表面之DNA適體連接UCNP,以980 nm紅外光激發UCNP,由從UCNP中釋放之530 nm綠光使二硫化錫(能隙2.2 eV)半導體表面電子由價帶激發至導帶,產生光電流(photocurrent),作為生物感測之訊號源。又當目標分子與DNA適體結合,使適體構型開始摺疊,進而縮短UCNP到元件表面之距離,此時釋放出之綠光便能激發更多元件內之電子並產生更強之光電流,即可以在不受德拜屏蔽效應影響下,進行生物分子測量。我們利用此機制進行了鉀離子、赭麴毒素A及多巴胺三種分子之生物感測,三種分子之生物感測以10X NMG緩衝液、及1X PBS緩衝液等高鹽溶液環境中測量,實驗結果顯示三種分子之感測皆能收到明顯之光電流訊號,並於高濃度下達到訊號飽和,且偵測極限皆可達皮莫爾濃度(pM)等級。我們亦利用朗謬耳吸附方程式(Langmuir isotherm adsorption model)計算三種分子所應之DNA適體之解離係數(Kd),求得三種適體之Kd值分別為1.2 μM、64.8 nM及307 nM,三者皆接近文獻值。最後我們將感測之訊號數值帶入簡易DNA適體長度計算模型,利用適體於接收目標分子後之長度變化會反應在電流訊號上之特性,進而回推出摺疊後之DNA適體長度,結果顯示折疊後鉀離子適體長度為2.44 ± 0.04 nm、OTA適體為3.84 ± 0.03 nm。至此,我們利用光電傳感機制成功於高鹽環境下量得生物訊號,突破德拜屏蔽效應之限制,未來期望此機制能應用於高鹽環境之人類檢體,如血液或尿液,之生物目標分子偵測及定量相關研究。 Field-effect transistors (FETs) have been widely applied in the field of biosensing detections with their excellent features, such as high sensitivity and real-time detection. When charged target molecules approach a FET device, the electric field exerted from the target molecules can enhance or suppress the number of charge carriers in the conducting channel of the FET device, thus alternating the channel current. However, when a FET biosensor is applied in high salt buffer solution, the ions in the solution screen the electric field coming out from the target molecules, then attenuating the electric signals generated in the FET biosensor. This phenomenon is known as the Debye-Hückel screening effect. Therefore, it is desirable to invent a brand new method to overcome the Debye-Hückel screening effect when using FET biosensors for detecting biomarkers in high salt buffers. Here we designed an optoelectronic device as a new biosensor, which is composed of three parts: a tin disulfide field-effect transistor (SnS2-FET), a single-stranded DNA aptamer, and upconverting nanoparticles (UCNPs). With one end of the aptamer modified on the surface of a SnS2-FET device, the other end of the aptamer was immobilized with a UCNP. The UCNP absorbs 980 nm radiation and emits 530 nm light, of which the green light at 530 nm can excite the charge carriers (i.e., electrons) in the conducting channel of SnS2 nanosheets (with a bandgap of ~2.2 eV) in a SnS2-FET to generate photocurrent. When the target molecules bind the aptamers, the aptamers fold and shorten the distance between UCNPs and the SnS2-FET, consequently intensifying the photocurrent inside the SnS2-FET. The increase of the photocurrent depends on the amount of target molecules captured by the aptamers. The photocurrent generated as a signal in the SnS2-FET relies on the photons, rather than electric field, coming out from UCNPs and penetrating the buffer solution used in the biosensing measurements. The penetrating capability of photons is expected to overcome the Debye-Hückel screening in high salt buffers. We have applied this optoelectronic biosensor to detect (a) potassium ions in 10 NMG, (b) ochratoxin A in 10X NMG, and (c) dopamine molecules in 1X PBS. Our results show that a clear photocurrent increment was obtained in each of these three experiments as the concentration of target molecules increased. Besides, we determined the dissociation constant (Kd) of the target-aptamer complex by a least-squares fit of the experimental data to the Langmuir isotherm adsorption model. The Kd values are 1.2 μM for potassium ion, 64.8 nM for ochratoxin A, and 307 nM for dopamine, respectively, with their specific aptamers. Moreover, we proposed a simple model to estimate the lengths of the folded aptamers used in this study. The results show that the lengths of the folded aptamers are 2.44 ± 0.04 nm for the potassium ion-specific aptamer and 3.84 ± 0.03 nm for the ochratoxin A-specific aptamer. In conclusion, we have been able to detect three targets in high salt buffer solutions, of which the detections are beyond the Debye-Hückel screening effect. In the future, this optoelectronic biosensor will be applied for detecting biomarkers in physiological fluids, such as serum or human whole blood, for practical clinical diagnosis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/54695 |
DOI: | 10.6342/NTU202002228 |
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顯示於系所單位: | 化學系 |
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