開發奈米預濃縮與週期性奈米金屬閘表面電漿共振感測器結合於免標定光學免疫分析平台

Abstract

在現今免疫分析方法中，要做到免標定的超低濃度檢測是目前多數生物感測平台所面臨的一大困難。雖然預濃縮方法可以有效地降低檢測濃度，但現今搭配預濃縮的免疫分析方法皆需要以螢光標記。螢光標記方法不僅使檢測流程較為繁複，且分析上較為困難。我們團隊於 2013 年成功地提出了以迴路電流表現觀察微奈米濃縮現象的方法，藉由此方法我們可以從電流圖表現分辨濃縮是否發生以及發生的位置。因為這個電流檢測技術讓我們離免標定免疫分析和預濃縮的結合更加接近。在本文中，我們進一步成功地結合了微奈米預濃縮機制與奈米金屬週期性表面電漿共振的光學免疫分析到一個微小化平台上，進而實現了全程免標定的可濃縮免疫分析平台。 免標定的奈米金屬週期性表面電漿共振檢測有許多優點，例如：整體元件體積小、不需特定入射光角度以及更高的表面靈敏度。然而，現今免標定的奈米金屬週期性表面電漿共振檢測的檢測極限，僅有到 ng/ml 的大小而已。因此，我們將免標定的奈米金屬週期性表面電漿共振檢測技術結合微奈米預濃縮機制，以讓檢測極限可以到達更低的水平（pg/ml）。 本研究使用導電性光阻在玻璃基板上以電子束微影方式製作出奈米金屬週期性結構 ( 週期 ~ 550 nm )，藉由傳統黃光製程以及軟微影製程做出以聚二甲基矽氧烷 ( Polydimethylsiloxane, PDMS ) 為基材的微流道，以奈米多孔性材料Nafion 實現奈米流道並對準於玻璃基板上之表面電漿檢測晶片旁，以完成微奈米預濃縮的結構。最後藉由氧電漿結合，將玻璃基板和 PDMS 基板結合( 共價鍵 )，以完成全程免標定的可濃縮免疫分析平台。 文中將討論，在整合兩項技術時所遇到的困難，以及對應不同檢測需求 時，可以改變的晶片設計參數。進而提出整個免疫分析平台在製作時整合的條件以及相對應的解決辦法。文末也分析了在使用此免疫分析平台時，所遇到的異常現象及可能原因。 在可濃縮的免疫分析平台的驗證上，我們使用牛血清 (Bovine Serum Albumin, BSA) 蛋白來做免疫分析，藉由通入 20 ng/ml 的牛血清抗體於濃縮流道中(10 min)，觀察有濃縮與沒有濃縮的共振光譜紅移現象。我們得到在沒有濃縮的晶片上，共振波長的紅移量僅有 0.42 nm，而有濃縮晶片共振波長的紅移量為5.33 nm。考慮量測好的牛血清抗體之參考光譜特性，我們發現濃縮區塊的濃度約為 200 µg/ml，進而推得濃縮倍率約為 10000 倍，且推出理論最低可以檢測濃度為 2 pg/ml。 總的來說，本論文表述了首次成功結合兩個技術於一個平台上並且成功運作的成果。在此之前，增強表面電漿共振感測器訊號的方法皆需要耗時的前處理，如金奈米粒子修飾。除此之外，本實驗也是首次利用表面電漿共振的訊號來驗證預濃縮技術的濃縮倍率，並且驗證奈米預濃縮的濃縮倍率可達一萬倍。在此可預濃縮的光學免疫分析平台上，我們搭配一自組的配電系統與光學系統即可以完成全程免標定的超低濃度目標生化物質檢測。

In the field of bio microelectromechanical systems (bio MEMS), detection of the low-abundance analytes without labelling is challenging because of difficulties of integration of preconcentration and label free sensing. Previously, an electrokinetic trapping (EKT)-based nanofluidic preconcentrator had been reported for providing a million-fold concentration factors that enable the validation of concentration process and the detection of trace and fluorescence-labelled analytes. However, the use of fluorescence-labelled analytes has suffered several disadvantages, e.g., additional sample preparation in an experimental workflow, high cost of labeling reagents, and difficulty in analyzing trace analytes. To monitor the concentration process without labelling, our group has presented a real-time dual loop electric current measurement system for label-free EKT-based nanofluidic preconcentrator. In this work, we further demonstrated a label-free biosensing platform by integrating a label-free nanofluidic preconcentrator with label-free surface plasma resonance(SPR) sensors. Bio molecular sample preconcentration was realized by a preconcentrator consisted of two microchannels, a concentration channel and a buffer channel, cast in Polydimethylsiloxane (PDMS) and a porous membrane (Nafion). The nanograting SPR sensor was fabricated by e-beam lithography, e-gun evaporation followed by the lift-off process. After glass-based SPR sensors and PDMS microchannels were fabricated, we patterned Nafion membrane at a specific position adjacent to the SPR sensor by using a microflow patterning method. Finally, PDMS-based microchannels were bonded to a glass patterned with Nafion and two square SPR sensors via bonding technique with oxygen plasma treatment. Recently, a 20 ng/ml Bovine serum albumin (BSA) in PBS was pumped into the platform, and was detected by SPR sensor with a red-shifted value of 0.42 nm. After ten minutes of preconcentration, 20 ng/ml BSA in PBS was detected with a red-shifted value of 5.33 nm. Comparing the references of the red-shifted values at different concentrations of BSA established in advance, the red-shifted value (5 nm) of 20 ng/ml BSA in PBS after preconcentration is the same as the red-shifted value of 200 μg/ml BSA in PBS. Hence, the preconcentration factor in this label-free platform was then determined to be approximately 10000 fold. In summary, a label-free immunoassay platform combining a preconcentrator which can improve the sensitivity limit by about 10000 fold with highly sensitive SPR sensors is realized. With a simple electrical and optical, low abundance analytes can be preconcentrated and sensed by this label-free platform.