以白光傅立葉轉換法偵測光學生物感測器陣列

Detection of optical biosensing arrays using white light Fourier transform method

林奕昌(Yi-Chang Lin)

指導教授 ： 蔡宛卲

國立暨南國際大學/科技學院/應用材料及光電工程學系/碩士(2014年)

https://doi.org/10.6837/NCNU.2014.00339

摘要

近年來，表面電漿共振技術已廣泛被應用在光學感測。生物光學感測器是利用光在不同環境中的共振或傳播行為，來測定不同生物細胞分子間的鍵結變化。傳統使用表面電漿型的光學生物感測器，必須外加稜鏡將光耦合至感測器中，且需要複雜且精準的操作，來達成表面電漿波與入射光的相位匹配。這幾年研究指出，週期性金屬奈米結構可以讓光激發表面電漿子，不需使用稜鏡即可將光耦合，故本研究採用晶片型奈米金狹縫陣列結構來做為感測器使用。先將待感測物質置於週期性奈米金狹縫陣列上，再以入射TM極化光打在週期性奈米金狹縫陣列結構，產生表面電漿波。量測穿透光譜，分析共振波長或強度變化來達到感測生物細胞分子之間交互作用的目的。奈米金狹縫陣列設計成二維矩陣方式，金膜陣列週期為500 nm，結構面積為5 mm×5 mm，狹縫深度為150 nm，狹縫寬度為60 nm，樣品利用外加壓克力的方式封裝成微流道，使注入溶液時的環境條件穩定。

實驗量測架設為一明場顯微鏡，結合以麥克森干涉儀為基礎的白光傅氏光譜儀來做為量測架設系統。光打入麥克森干涉儀後，光程差由壓電位移元件控制在0至35 μm之間。由光程差所形成的干涉條紋使用CCD(charge-coupled device)攝影機收光，並以數學軟體MatlabR做傅氏分析轉換成頻譜。本研究將奈米金狹縫陣列放入不同濃度甘油與水混合溶液中，藉以量測樣品在不同環境折射率下其穿透光譜變化。甘油與水混合溶液的折射率範圍為1.33至1.38。量測波長靈敏度，其定義為共振波長對應折射率變化。所得到的波長靈敏度數值為213 nm/RIU(refractive index unit)。因使用波長峰值對應折射率變化所得到的波長線性關係並不明顯，故本研究使用質心法來增加線性度，質心法靈敏度數值為130 nm/RIU。雖然靈敏度降低，但改善了波長線性度。此外量測強度靈敏度，其定義為強度對應折射率變化。所得到的強度靈敏度數值為99 %/RIU。為了提升感測強度靈敏度，使用多光譜積分法，得到的強度靈敏度數值為2078 %/RIU，感測強度靈敏度有明顯提升。此外，研究進行了牛血清白蛋白與牛蛋白抗體的結合的感測。利用此架設系統量測牛血清白蛋白與牛蛋白抗體的結合情形；為了提升感測靈敏度，分析上使用多光譜積分法來提高感測強度靈敏度。實驗中所能偵測的牛血清白蛋白濃度為6.06 μM，牛蛋白抗體濃度為60 nM。實驗結果顯示，藉由觀察光譜變化，可以容易看出牛血清白蛋白與牛蛋白抗體是否結合。
關鍵詞：表面電漿共振、白光傅氏光譜儀。

關鍵字

白光傅立葉轉換法 ； 光學生物感測器

並列摘要

In recent years, surface plasmon resonance technique has been widely used in optical sensing. Optical biosensors detect the binding energy between bio-molecules using the resonance or propagation behavior of light under different environments. Conventionally, the optical biosensor based on surface plasmon resonance needs an extra prism to couple light to the sensor. Complicated and accurate adjustment is required for phase matching between the surface plasmon wave and the incident light. In recent years, some research works have shown that surface plasmon wave can be excited when light passing through the periodic metallic nanostructures directly, without coupling via a prism. In this study, the optical sensor is designed as a chip-based gold nanoslit array structure. A TM-polarized light is normally incident on the gold nanoslit array with the analyte on top of it. The surface plasmon wave is then excited on the gold surface. The transmission spectrum is measured to detect the interaction between sensing analytes through analyzing the resonance wavelength shifts or intensity changes of the spectrum. The gold nanoslit array is designed as a two-by-two matrix, with slit period 500nm, structure area 5 mm×5 mm, slit depth 150 nm, and the slit width 60 nm. The sample was encapsulated with acrylic as a microfluidic chip, which stabilizes the environment during liquid injection.

The experimental set up combines a bright field microscopy with a white-light Fourier spectrometer based on a Michelson interferometer system. In the Michelson interferometer system, the optical path difference is controlled between 0 to 35 μm. The interference pattern is recorded by a charge-coupled-device (CCD) camera as an interferogram. The interferogram is later transformed into spectrum by Fourier analysis using MatlabR software. In this work, the gold nanoslit array was covered by glycerin solution with different concentration. The refractive index of glycerin solution varies from 1.33 to 1.38. Transmission spectra under different environmental index are detected. The wavelength sensitivity, defined as resonance wavelength changes with refractive index, is measured as 213 nm/RIU (refractive index unit). Since the linearity of wavelength sensitivity is not good enough, we further use a center mass method to improve the linearity. The measured wavelength sensitivity under center mass method is 130 nm/RIU. Although the sensitivity is lower, a better linearity is obtained. Moreover, the intensity sensitivity, defined as intensity changes with refractive index, is measured as 99 %/RIU. To increase intensity sensitivity, a multispectral integration method is used. An increased sensitivity of 2078 %/RIU is obtained. Furthermore, the detection of the binding between bovine serum albumin (BSA) and bovine protein antibodies (anti-BSA) are measured, with BSA concentration 6.06 μM and anti-BSA 60 nM. The multispectral integration method is used to analyze the experimental results for better intensity sensitivity. Experimental results show that the binding between BSA and anti-BSA can be easily detected by the spectra change.
Key words：Surface plasmon resonance, White-light Fourier spectrometer

並列關鍵字

White Light Fourier Transform Method ； Optical Biosensing

參考文獻

[1] S. M. Borisov and O. S. Wolfbeis, "Optical Biosensors," Chem. Rev., vol. 108, pp. 423-461, 2008.

[2] 謝振傑, "光纖生物感測器," 物理雙月刊, pp. 704-710, 2006.

[3] Andrey L, Ghindilis, P. Atanasov, M. Wilkinst, and E. Wilkins, "Immunosensors electrochemical sensing and other engineering approaches," Biosens. Bioelectron. vol. 13, pp. 113-131, 1998.

[4] P. I. Reyes, C.-J. Ku, Z. Duan, Y. Lu, A. Solanki, and K.-B. Lee, "ZnO thin film transistor immunosensor with high sensitivity and selectivity," Appl., vol. 98, p. 173702, 2011.

[5] M. POHANKA and P. SKLADAL, "Piezoelectric immunosensor for the direct and rapid detection of Francisella tularensis," Folia Microbiol., vol. 52, pp. 325-330, 2007.