利用表面增強拉曼晶片建立一個檢測細胞膜上生物分子的平台

Abstract

細胞膜上的物質傳輸、識別和結合與各種生理機制密切相關。我們建立了一種基於支撐細胞膜的拉曼技術檢測平台來研究細胞膜上的事件。通過將細胞膜鋪設在具有拉曼增強結構的基材上，利用拉曼光譜檢測細胞膜及其相互作用的物質。由於細胞膜只有奈米級的厚度，上面只有少量的蛋白質和與其作用的物質，我們使用膠體粒子阻擋後鍍金形成的金三角結構晶片來增強訊號，並研究不同電漿製程參數對晶片結構的影響，以進一步改善晶片增強訊號的能力，也找到了適當的雷射參數以避免生物分子在實驗過程中被破壞。我們首先在增強晶片上分別檢測霍亂毒素次單元B (CTB) 、1,2-二油酰-sn-甘油-3-磷酸膽鹼(DOPC)脂雙層膜和巨大質膜囊泡膜片(GPMV patch)的訊號。實驗結果顯示，我們成功在增強晶片上獲得了霍亂毒素次單元 B 和 1,2-二油酰-sn-甘油-3-磷酸膽鹼脂雙層膜的訊號，這些訊號與標準品的拉曼訊號具有一致性。然而，巨大脂質膜囊泡膜片(GPMV patch)的訊號卻非常微弱，其特徵峰位置與標準品不一致。這可能是因為此研究的增強晶片是由膠體粒子阻擋後鍍金形成的金三角結構晶片，訊號增強顯著發生在約100 奈米寬的尖角隙縫區域。霍亂毒素次單元 B 的分子大小約為 100 奈米，能夠進入這些空隙，從而增強訊號。之前文獻也指出，1,2-二油酰-sn-甘油-3-磷酸膽鹼脂雙層膜能鋪於增強晶片底部，並進入增強區域，以造成訊號增強。然而，當20微米大小的巨大脂質膜囊泡鋪於增強基材形成膜片時，細胞膜的張力很可能會使膜片懸浮在金三角結構上，而難以落入尖角增強區域，從而導致訊號微弱。我們進一步分別使用 1,2-二油酰-sn-甘油-3-磷酸膽鹼脂雙層膜內加入的單唾液酸四己糖神經節苷脂(GM1)，以及巨大質膜囊泡膜片內的天然受體來捕捉霍亂毒素次單元B 至膜片上，以了解這些膜片配合目前拉曼增強基材能否做為檢測器的可能性。經由實驗組和控制組相扣處理後的訊號顯示，在 1,2-二油酰-sn-甘油-3-磷酸膽鹼脂雙層膜上捕捉到的霍亂毒素次單元 B 可被檢測到，但由巨大質膜囊泡膜片捕捉到的霍亂毒素次單元 B 的訊號則是微弱難以辨別。這結果也符合我們對於 1,2-二油酰-sn-甘油-3-磷酸膽鹼脂雙層膜和巨大質膜囊泡膜片所坐落的位置的推測，當霍亂毒素次單元 B 黏到 1,2-二油酰-sn-甘油-3-磷酸膽鹼脂雙層膜上時，霍亂毒素次單元 B仍能位於金三角尖角空隙之間，但當霍亂毒素次單元 B 黏到巨大脂質膜囊泡膜片上時，霍亂毒素次單元 B 則會遠離訊號增強區域，導致其無法被偵測到。

The transport, recognition, and binding of substances on cell membranes are closely related to various physiological mechanisms. We established a detection platform based on Raman technology with supported cell membranes to study events on cell membranes. By depositing cell membranes on substrates with Raman-enhanced structures, we used Raman spectroscopy to detect cell membranes and their interacting substances. Since cell membranes are only nanometer-thick, with only a small number of proteins and interacting substances on them, we used gold nanotriangle structure chips formed by colloidal lithography to enhance the signal. We studied the impact of different plasma processing parameters on the chip structure to further improve the chip's signal enhancement capability, and also found appropriate laser parameters to avoid damaging biomolecules during the experiment. We first detected the signals of cholera toxin subunit B (CTB), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer, and giant plasma membrane vesicle (GPMV) patches on the enhancement chip. The experimental results showed that we successfully obtained the signals of CTB and DOPC lipid bilayer on the enhancement chip, which were consistent with the Raman signals of the standards. However, the signal of the GPMV patch was very weak, and its characteristic peak position was inconsistent with the standard. This could be because the enhancement chip used in this study was a gold nanotriangle structure chip, with significant signal enhancement occurring in the narrow gaps of about 100 nanometers. The size of the CTB molecule is about few nanometers, allowing it to enter these gaps and thus enhance the signal. Previous literature has also pointed out that the DOPC lipid bilayer can form at the bottom of the enhancement chip and enter the enhancement area to cause signal enhancement. However, when the 20-micron-sized GPMV forms a patch on the enhancement substrate, the membrane tension is likely to suspend the patch on the gold triangular structure, making it difficult to fall into the narrow enhancement area, resulting in a weak signal. We further used DOPC lipid bilayers with monosialotetrahexosylganglioside (GM1) and GPMV patches with natural receptors to capture CTB onto the patches to explore the potential of these patches as sensors. The signal processed by experimental and control groups showed that CTB captured on DOPC lipid bilayer could be detected, but the signal of CTB captured by the GPMV patch was weak and difficult to distinguish. This result also aligns with our hypothesis about the positions of the DOPC lipid bilayer and GPMV patch. When CTB adheres to the 1,2-dioleoyl-sn-glycero-3-phosphocholine lipid bilayer, it remains in the gap between the gold triangular tips, but when it adheres to the GPMV patch, CTB could be far from the signal enhancement area, making it undetectable in our current chip.