乳癌細胞中胺基酸與生物胺之微胞電動力毛細管層析分析

Abstract

本論文之主要目的為發展微胞電動力毛細管層析(micellar electrokinetic chromatography, MEKC)結合發光二極體誘導螢光偵測法(light-emitting diode-induced fluorescence detection)分析經naphthalene-2,3-dicarboxaldehyde (NDA)衍生化的胺基酸(amino acids)以及生物胺(biogenic amines)。藉由探討緩衝液的pH值及所含之十二烷基硫酸鈉(sodium dodecyl sulfate, SDS)、聚氧化乙烯(poly(ethylene oxide), PEO)和Tris-borate (TB)濃度對分離效率與解析度之影響，發現最佳化的分離條件為：用含40 mM SDS的0.5 M TB (pH 10.2)緩衝溶液來填充毛細管，及使用含35 mM SDS之100 mM TB (pH 9.0)緩衝溶液來配製0.1% PEO。在此條件下，我們可於16分鐘內成功分離14種胺類小分子，其偵測極限(S/N = 3)從2.1 nM甘胺酸(glycine, Gly)到19.2 nM血清素(serotonin, 5-HT)。分析物遷移時間的相對標準偏差(relative standard deviation, RSD)皆小於0.5% (n = 5)。應用此分離技術，分別對乳癌細胞(MCF-7)與正常乳房表皮細胞(H184B5F5/M10) (2-3 × 106 cells/mL)進行分析(三次測量)，並定量細胞中9種(MCF-7)以及10種(H184B5F5/M10)胺基酸，包括絲胺酸(serine, Ser)、麩醯胺酸(glutamine, Gln)、組胺酸(histidine, His)、Gly、丙胺酸(alanine, Ala)、牛磺酸(taurine, Tau)、酪胺酸(tyrosine, Tyr)、麩胺酸(glutamate, Glu)、纈胺酸(valine, Val)、天冬胺酸(aspartate, Asp)。發現兩種細胞在部分胺基酸含量上確實有差異，推測部分原因與癌細胞需要大量分化生殖，消耗能量有關。我們也於細胞繼代後不同時間，收集細胞去做胺基酸分析，並計算單一細胞胺基酸的含量，癌細胞由於生長速率較快，經過48及72小時後，觀察到部份胺基酸是明顯減少的，反觀正常細胞，則因為生長速度緩慢，胺基酸消耗的速度較不明顯。 為了提高偵測極限，我們利用相同的緩衝溶液條件與毛細管處理方式，進行線上濃縮與分離胺基酸與生物胺。在電壓驅動下，經NDA衍生化的分析物進入到含有SDS的PEO溶液當中，會因樣品區帶和PEO溶液之間電導度與黏度的差異，進行線上濃縮與分離。藉由探討樣品區帶中TB的濃度和添加氯化鈉(NaCl)的量對濃縮效果的影響，發現樣品濃縮倍率可由4.4倍(His)至38.7倍(Glu)，其偵測極限(S/N = 3)從0.10 nM (Glu)到3.63 nM (5-HT)。利用此線上濃縮與分離技術，分析0.284-μL 之乳癌細胞(MCF-7) (2 × 105 cells/mL)，並定量細胞中9 種胺基酸，得到的結果與前一部份使用單純分離的方法是相近的。

We have demonstrated separation of the derivatives of amino acids and biogenic amines with naphthalene-2,3-dicarboxaldehyde (NDA) by micellar electrokinetic chromatography (MEKC) in conjunction with a purple light-emitting diode-induced fluorescence detection using poly(ethylene oxide) (PEO). The roles that the concentrations of PEO, sodium dodecyl sulfate (SDS), and Tris-borate (TB) at various pH values played in resolution and separation efficiency of the analytes have been investigated. After injecting the sample to a capillary filled with 0.5 M TB buffer (pH 10.2) containing 40 mM SDS, 0.1% PEO that has been prepared in 100 mM TB (pH 9.0) containing 35 mM SDS enters the capillary. This MEKC condition allows separation of 14 analytes within 16 minutes, with limits of detection (LODs) at signal-to-noise ratio (S/N) of 3 ranging from 2.06 nM (glycine, Gly) to 19.2 nM (serotonin, 5-HT). RSD values (n = 5) of the migration times for the analytes are all less than 0.5%. This approach has been validated for the analysis of human breast cancer cells (MCF-7) and normal epithelial cell line (H184B5F5/M10) (2-3 × 106 cells /mL). The amounts of 9 (MCF-7) or 10 (H184B5F5/M10) amino acids, including serine (Ser), glutamine (Gln), histidine (His), Gly, alanine (Ala), taurine (Tau), tyrosine (Tyr), glutamate (Glu), valine (Val), and aspartate (Asp) in one cell could be calculated, respectively. The amounts of some amino acids in MCF-7 and H184B5F5/M10 are significantly different, maybe because of proliferation of cancer cells which consume much energy. Besides, we calculated the amounts of amino acids in cells incubated at different time interval, and found that the consumption of some amino acids in cancer cells rather than normal cells was obvious. In order to further improve the sensitivity of the amino acids and biogenic amines, we developed an on-line concentration technique using similar MEKC conditions. Once high voltage is applied, the NDA derivatives enter PEO solution containing SDS and then become stacked due to decreases in electric field and increases in viscosity. We have also found that TB and sodium chloride in the sample zone play some roles in determining the stacking efficiency. This on-line concentration MEKC technique allows improvement in the detection of the analytes by factors ranging from 4.4 (His) to 38.7 (Glu), leading to their LODs ranging from 0.10 nM (Glu) to 3.63 nM (5-HT). This approach has been validated by the analysis of 0.284-μL MCF-7 cells (2 × 105 cells/mL), and the results were consistent with those described in the first part.