應用光電傳感生物感測技術突破德拜屏障長度於高鹽緩衝液中偵測生物指標分子

An Optoelectronic Biosensor for Detecting Biomarkers in High Salt Buffers beyond Debye Screening Length

何長榮(Chang-Rong Ho)

指導教授 ： 陳逸聰

國立臺灣大學/理學院/化學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201902509

摘要

場效電晶體(field-effect transistor, FET) 應用在生物感測上具有高靈敏度、即時性等優勢，然而所要感測的生物分子不易在高鹽類濃度的環境下進行檢測，如人體血液和尿液。因為環境溶液中的高濃度的離子，會屏蔽待測分子給予偵測通道的作用電場(interacting electric field)，此現象稱為德拜屏蔽效應(Debye screening effect)。這是傳統場效電晶體生物感測器要應用在臨床診斷所面臨的重大限制之一，本研究目的即是要突破長久以來Debye screening effect對場效電晶體生物感測器的限制。
本研究發展出新穎的光電生物感測技術(optoelectronic biosensor)，可以在高鹽濃度的緩衝液中(例如：10× NMG溶入20 mM Ca2+，其Debye length(λD) ~ 0.61 nm) 感測到不同濃度的生物指標分子。此項技術有別於傳統之場效電晶體由外在電場誘發帶電載子的機制，而是利用光激發半導體元件的帶電載子，因此可突破Debye screening effect的限制。光電生物感測技術包含三個主要組件：二維奈米級半導體材料二硫化錫-場效電晶體元件(SnS2-FET)、上轉換奈米粒子(upconverting nanoparticles, UCNPs)與高選擇性DNA適體(specific aptamer, Apt)。以 SnS2-FET作為感應電子元件(electronics)；UCNPs作為光來源(optics)；DNA適體則有兩個重要功用：一為修飾於SnS2-FET作為生物指標分子的專一性受體(receptor)，二為其兩端分別鍵結SnS2-FET與UCNPs成為UCNP/Apt/SnS2-FET系統之光電生物感測元件，並且能藉由與生物指標分子的專一結合而自身折疊收縮，控制UCNPs與SnS2-FET元件的距離。二硫化錫為n-型半導體具有約2.2 eV（~560 nm）的能隙，當UCNPs吸收980 nm的雷射紅外光後，會釋放530 nm的綠光，進而激發SnS2-FET產生光致電流(photocurrent)。當生物指標分子進入UCNP/Apt/SnS2-FET感測元件系統時，適體將與之結合而折疊，縮短了UCNP和SnS2間的距離，SnS2-FET感受到的530 nm光強度因而上升，使SnS2-FET被激發的電子數目增加，故我們可藉由SnS2-FET元件的光致電流訊號變化來感測生物指標分子，並定量生物指標分子的濃度。
本研究利用UCNP/Apt/SnS2-FET感測系統檢測兩項具重要生理意義的生物指標分子：鉀離子(potassium ions)、赭麴毒素(ochratoxin A, OTA)，並可成功於pH 7.4 之高鹽環境10× NMG(λD ~ 0.69 nm)、 10× NMG溶入20 mM Ca2+緩衝溶液(λD ~ 0.61 nm)下分別感測到不同濃度的鉀離子、赭麴毒素。鉀離子感測的線性工作範圍為10-10 ~ 2.5×10-4 M，偵測極限達100 pM；赭麴毒素感測的線性工作範圍為10-11 ~ 10-4 M，偵測極限達10 pM。另外，我們提出DNA適體與生物指標分子結合後之伸縮長度簡易模型公式，計算出鉀離子適體與K+專一結合後的折疊長度為 = 2.54 ± 0.26 nm；赭麴毒素適體與OTA專一結合後的折疊長度為 = 3.48 ± 0.29 nm。UCNP/Apt/SnS2-FET感測技術突破傳統場效電晶體之Debye screening effect，未來於醫學臨床上人體血液或尿液之即時分析與應用具有極大的潛能。

關鍵字

二硫化錫 ； 上轉換奈米粒子 ； 生物感測器 ； 德拜屏蔽效應 ； 適體 ； 場效電晶體

並列摘要

Field-effect transistors (FETs) have long been employed as high-sensitive and real-time biosensors for detecting biomarkers, but the biomolecules to be sensed are not easily detected in high salt concentration environments, such as human serum and urine. Because the high concentration of ions and charged molecules in the solution shields the interacting potential exerted by the charged targets toward the sensing channel of an FET, the signals are severely attenuated. This phenomenon is called Debye screening effect. This is one of the major limitations of traditional field effect transistor biosensors for clinical diagnosis. The purpose of this study is to overcome the crucial limitations of the long-term Debye screening effect on field-effect transistor biosensors.
We developed a novel optoelectronic biosensor that can detect different concentrations of targeted biomarkers in high salt buffers, like 10× NMG with 20 mM Ca2+(Debye length ~ 0.61 nm. The working principle is quite different from the traditional field effect transistor which induces a charge carrier by external electric field, but uses laser light to activate the charge carrier in the semiconductor channel of FETs. Therefore, our optoelectronic biosensor can break through the limitations of the Debye screening effect. The optoelectronic biosensor is composed of a 2D layered semiconducting nanosheets SnS2-FET in conjunction with upconverting nanoparticles (UCNPs) via aptamer linkers (referred to as UCNP/Apt/SnS2-FET). The aptamer has two roles in a UCNP/Apt/SnS2-FET biosensor; first, it serves as a receptor with specific binding to a selected target, and second, it controls the distance between UCNP and SnS2-FET (denoted by ΔUCNP-FET). Upon 980 nm excitation, the immobilized UCNP emits ~530 nm green light to excite the n-type SnS2 nanosheets (with the band gap of ~2.2 eV, corresponding to ~ 560 nm light)-FET to generate photocurrent. Without target binding, the aptamer is in its free form, making a longer ΔUCNP-FET and weaker UCNP-illumination on the SnS2-FET; comparatively after the target-aptamer binding, a shorter ΔUCNP-FET due to the folded aptamer will result in stronger UCNP-illumination on the SnS2-FET. Therefore, upon target binding, the photocurrent of the SnS2-FET increases. Based on the photocurrent signal change in the UCNP/Apt/SnS2-FET, targeted biomarkers can be detected and quantified.
The aim of this research is to investigate the feasibility, sensitivity, and detection limit of a UCNP/Apt/SnS2-FET biosensor by detecting two targeted biomolecules: potassium ions and ochratoxin (OTA). The experimental results demonstrate that we can successfully detect potassium ions and OTA in 10× NMG buffer(λD ~ 0.69 nm) and 10× NMG buffer with 20 mM Ca2+(λD ~ 0.61 nm) at pH 7.4, respectively.The linear working range of potassium ions biosensing is 10-10 ~ 2.5×10-4 M, and the LOD is 100 pM; The linear working range of OTA biosensing is 10-11 ~ 10-4 M, and the LOD is 10 pM. In addition, we propose a simple model to estimate the length of the folded aptamer in the aptamer-target binding experiments by UCNP/Apt/SnS2-FET. The folding length of the potassium specific-aptamer binded with K+ is = 2.54 ± 0.26 nm. The folding length of the ochratoxin specific-aptamer binded with the OTA is = 3.48 ± 0.29 nm. UCNP/Apt/SnS2-FET breaks through the Debye screening effect of traditional field effect transistors and it has great potential for real-time analysis human blood or urine in medical clinical applications.

並列關鍵字

SnS2 ； upconverting nanoparticles(UCNPs) ； Biosensor ； Debye screening effect ； Aptamer ； Field-effect transistor (FET)

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