本研究利用奈米碳管 (carbon nanotube, CNT)，驗證具有高吸收係數與高散射能力之奈米材料，可用於標定待測物-人類血清白蛋白 (human serum albumin, HSA) 以提供光學訊號，製備出新穎之生物感測器，並利用紫外/可見光吸收光譜儀 (ultraviolet-visible absorption spectroscopy, UV-Vis) 來定量待測物-人類血清白蛋白之濃度。
本生物感測器由檢測試片 (sensing-substrate) 和標定物奈米碳管(CNT-label) 所組成。檢測試片主要作為提供專一鍵結待測物-人類血清白蛋白之反應載台，其製備乃利用3-氨丙基三乙氧基矽烷 (3-aminopropyltriethooxysilane, APTES) 進行玻璃之表面改質，使其具有生物相容性，而後依序固定上單株人類血清白蛋白抗體 (anti-human serum albumin antibody, monoclonal, AHSA) 與小牛血清蛋白 (bovine serum albumin, BSA)，即完成檢測試片的製備。而標定物奈米碳管則用以提供光學訊號，其製備方法為將帶有羧基 (carboxyl group, -COOH) 改質後之奈米碳管 (COOH-modified CNT)，利用1-(3-二甲氨基丙基)-3-乙基碳二甲胺 (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, EDC)、N-羥基丁二酰亞胺 (N-hydroxysuccinimide, NHS) 與多株人類血清白蛋白抗體 (anti-human serum albumin antibody, polyclonal, AHSA*) 反應，即完成能與待測物-人類血清白蛋白進行專一鍵結的標定物奈米碳管。
在生物感測器之操作上，先於檢測試片上加入待測之人類血清白蛋白溶液，而後再以標定物奈米碳管對待測物-人類血清白蛋白進行標定，便完成此生物感測器之最終結構，稱之為CNT-labeled sensing-substrate。接著利用紫外/可見光吸收光譜儀，量測不同人類血清白蛋白濃度鍵結之CNT-labeled sensing-substrates穿透率，並加以量化。結果顯示，當待測物-人類血清白蛋白濃度越高，生物感測器之穿透率越低。且在2 × 10-1 mg/ml - 2 × 10-5 mg/ml之人類血清白蛋白濃度間，生物感測器之穿透率與人類血清白蛋白濃度之對數 (log) 呈現線性關係，此生物感測器之偵測極限 (detection limit) 可達2.88 × 10-5 mg/ml。
本研究成功驗證以高吸收係數和高散射能力之奈米碳管，可作為標定物，製作新穎生物感測器，並以紫外/可見光吸收光譜儀當作檢測儀器之可行性。和同樣使用紫外/可見光吸收光譜儀當作檢測儀器之生物感測器相比，本研究之特點如下：使用新穎之機制即藉由使用奈米碳管來當作提供光學訊號之材料，比起以往使用金奈米粒子之生物感測器，可達低檢測極限 (2.88 × 10-5 mg/ml)，且具有更低的材料成本 (商用奈米碳管的價錢遠低於商用金奈米粒子)、較大之檢測範圍 (可達0.43-3000 nM)。此外，本研究所製備之生物感測器，在不同人類血清白蛋白濃度下，已經能以肉眼辨識顏色之不同，且在腎臟疾病檢測上具有指標性意義。因此，未來在蛋白質檢測上將具有極大的應用潛力。

In this study, a novel biosensor was developed based on high absorption and high scattering ability nanostructured materials to enhance the optical signal. Carbon nanotubes (CNTs) were used as a label material to demonstrate the feasibility for this approach. Human serum albumin (HSA) was selected as a detection target for its importance as a biomarker of liver function. The biosensor for HSA detection was characterized by ultraviolet-visible absorption spectroscopy (UV-Vis).
The biosensor is composed of two components, a sensing-substrate and a CNT-label. The sensing-substrate is made of a piece of glass with the surface modified by bovine serum albumin (BSA) / anti-human serum albumin, monoclonal (AHSA) / 3-amicopropltriethoxysilane (APTES), to provide specific binding to HSA. The CNT-label is composed of CNTs which were immobilized with anti-human serum albumin, polyclonal (AHSA*) to label the detected HSA on the sensing substrate. AHSA* was covalently bound on CNTs with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) reaction to fabricate the CNT-label.
The detection procedure of this biosensor was described as following. First, the sensing-substrate was immersed in HSA solution for the specific bonding of HSA on sensing-substrate. Second, the HSA-bonded sensing-substrate was immersed in a AHSA* modified CNT-label solution for the bonding of CNT-label with the detected HSA on the sensing-substrate (CNT-labeled sensing substrate). Finally, the optical transmission was measured by UV-Vis.
The results show that there is a consistent reduction of transmission with increasing HSA concentrations. The reduction of the optical transmission is mainly contributed by CNTs bound on the substrates because of their high absorption coefficient and the high scattering ability. Moreover, the calibration results show good linearity between HSA concentration in log scale and reduction in optical transmission. The biosensor exhibits high sensitivity for the HSA detecting with a detection limit of 2.88 × 10−5 mg/ml.
Compared with other nanomaterial-based immunoassay biosensors which also use UV-Vis for signal measuring, this biosensor exhibits lower detection limit (2.88 × 10−5 mg/ml) and wider detection range (0.43-3000 nM) than those using gold nanoparticle for antigen detection biosensors. Moreover, this approach provides an innovative mechanism to detect antigen. Above results demonstrate the feasibility of using nanostructured material with high absorption and high scattering ability as a label material for biosensors that can quantify antigen concentration, and provide high-sensitivity and wide detection range applications.

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