利用奈米壓印技術整合奈米預濃縮機制與週期性奈米狹縫表面電漿共振感測器於免標定免疫分析平台

Preconcentrator Integrated a Periodic Metallic Nanoslit-based Surface Plasmon Resonance (SPR) Sensor Using Nanoimprinting Lithography for Immunoassay

李國翰(Guo-Han Lee)

指導教授 ： 沈弘俊

國立臺灣大學/工學院/應用力學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201601954

摘要

本研究成功開發一新型檢測平台，藉由奈米壓印技術(Nanoimprint Lithography)整合奈米流體預濃縮機制(Nanofluidic Preconcentration Mechanism)與週期性奈米金屬表面電漿共振(Periodic Metallic Surface Plasmon Resonance)感測器。藉由使用微奈米流體預濃縮法將待測活性物質進行濃縮，再以電位差控制將濃縮區塊限制在表面電漿共振感測晶片上，藉由光譜訊號紅移(Red-shift)進行免疫分析。
首先，用電子束微影(E-beam Lithography)製程和反應離子蝕刻(Reactive Ion Etching)技術在矽晶圓基材上定義週期性奈米狹縫結構。以環烯烴類聚合物(Cyclic Olefin Polymer, COP)作為實驗室晶片的基板材料，與矽晶圓母模進行奈米熱壓印將奈米結構轉印至高分子聚合物上，經由遮罩以濺鍍機完成局部鍍金製程，再使用奈米多孔性材料Nafion作為奈米流道並對準於聚合物基板上的表面電漿共振晶片之間，確立奈米流體預濃縮的結構。再藉由傳統黃光微影製程和軟微影(Soft Lithography)製程製作以聚二甲基矽氧烷(Polydimethylsiloxane, PDMS)為材料的微流道。最後經由化學表面修飾後以氧電漿結合高分子聚合物基板和微流道，以完成可預濃縮免標定免疫分析晶片。
我們所使用之生物檢測樣本為牛血清(Bovine Serum Albumin, BSA)蛋白，其所帶有的雙硫鍵可以直接與金進行共價鍵結。通入20 ng/mL的牛血清抗體進入濃縮流道內，藉由操控電壓使濃縮發生在表面電漿共振晶片上，接者比對實驗控制組與對照組的穿透特性光譜，經由紅移(Red Shift)量的差異和牛血清抗體濃度參考曲線，我們發現濃縮將20 ng/mL抗體濃度提升到大約200 µg/mL，濃縮倍率達到10,000倍，藉此可以推論最低檢測極限約為2 pg/mL。
綜觀而論，奈米壓印技術成功達到量產(Mass Production)、低材料成本(Low-cost)和低時間成本的快速製程，且奈米流體預濃縮降低了檢測下限，加上表面電漿共振具有高靈敏度(High Sensitivity)、即時(Real-time)檢測和免標定(Label-free)的優勢，我們藉由簡便的量測系統完成了一個免標定、微量的超低濃度檢測平台。

關鍵字

奈米壓印技術 ； 電驅動奈米流體預濃縮 ； 週期性奈米金屬表面電漿共振 ； 生物感測器 ； 免標定免疫分析 ； 實驗室晶片

並列摘要

In this study, a high sensitivity biosensing platform by integrating nanofluidic preconcentrator with periodic metallic surface plasmon resonance sensor has been developed. The target protein, was concentrated to enhance sensitivity of immunoassay. Firstly, the concentrated protein plug was trap in the sensing area of slit-based Surface Plasmon Resonance (SPR) in a microfluidic channel by electrical potential difference. The antibody-antigen interaction on periodic metallic slits resulted in, a red-shift of the resonant spectrum signals which is corresponding to the numbers of the antibody-antigen conjugation.
The periodic nano-grating structure were clarified and fabricated on a silicon wafer as a mold by E-beam lithography and reactive ion etching. The nanostructure was transferred onto a cyclic olefin polymer (COP) by nanoimprint lithography. The gold was deposited on the grating structure of COP by sputter. A porous material, Nafion, was used as the ion-selective channel and, was aligned to the metallic grating structure on COP. Microchannels were made by polydimethylsiloxane (PDMS) using soft lithography process. After the chemically modified surface treatment, the COP can be bond with PDMS by oxygen plasma. The metallic SPR sensor was located in one of parallel microfluidic channels, which were located cross over the Nafion channel.
In this study, bovine serum albumin (BSA) and anti-BSA were used as the testing samples. The disulfide bonds on it would form covalent bonds with Au and is able to be detected by spectrometer. Thereafter, 20 ng/mL of BSA antibodies was introduced into the concentration channel. Then, by adjusting electrical potential, the antibodies were condensed on the surface plasmon resonance chip. The concentrated magnification can be sufficiently derived by the red-shifted value deference and the reference curves of BSA antibody concentration. In summary, the concentration fold can be raised up to approximately 10,000 folds as the 20 ng/mL of antibodies was condensed to 200 µg/mL. Thus, we can conclude that the minimal detectable limit is about 2 pg/mL.
In conclusion, the nanoimprint lithography has several advantages, such as, mass production, low cost, and time saving. Furthermore, the preconcentrator has significantly broken through regarding the detectable limit and SPR has the advantages of high sensitivity, real-time detection, and label-free features. By integrating these two techniques, we can have a label-free, micro-concentration detectable platform by using simple measurement system.

並列關鍵字

Nanoimprint Lithography (NIL) ； Electrokinetic-based Nanofluidic Preconcentration ； Periodic Metallic Nanoslit-based Surface Plasmon Resonance (SPR) ； Biosensor ； Label-free Immunoassay ； Lab on a chip

參考文獻

[2] A. Hoos and C. Cordon-Cardo, Tissue microarray profiling of cancer specimens and cell lines: opportunities and limitations. Laboratory investigation, 2001. 81(10): p. 1331-1338.

[3] J.C. Mills, K.A. Roth, R.L. Cagan, and J.I. Gordon, DNA microarrays and beyond: completing the journey from tissue to cell. Nature Cell Biology, 2001. 3(8): p. E175-E178.

[4] A. Sassolas, B.D. Leca-Bouvier, and L.J. Blum, DNA biosensors and microarrays. Chemical reviews, 2008. 108(1): p. 109-139.

[5] K.-I. Chen, B.-R. Li, and Y.-T. Chen, Silicon nanowire field-effect transistor-based biosensors for biomedical diagnosis and cellular recording investigation. Nano Today, 2011. 6(2): p. 131-154.

[6] M.L. Kovarik, D.M. Ornoff, A.T. Melvin, N.C. Dobes, Y. Wang, A.J. Dickinson, P.C. Gach, P.K. Shah, and N.L. Allbritton, Micro total analysis systems: fundamental advances and applications in the laboratory, clinic, and field. Analytical chemistry, 2012. 85(2): p. 451-472.