基于美拉德反應(yīng)產(chǎn)物作為熒光和共振瑞利散射的傳感平臺(tái)快速檢測(cè)阿斯巴甜

趙艷梅，張小林，周 尚，楊季冬，，4*，高 佩

（1.重慶三峽學(xué)院 環(huán)境與化學(xué)工程學(xué)院，重慶 404100；2.東肯塔基大學(xué)化學(xué)系，美國(guó) 肯塔基州里士滿(mǎn) 40475；3.長(zhǎng)江師范學(xué)院 化學(xué)化工學(xué)院，重慶 408100；4.西南大學(xué) 化學(xué)化工學(xué)院，重慶 400715；5.佐治亞州佩恩學(xué)院 數(shù)學(xué)、科學(xué)與技術(shù)系，美國(guó)，佐治亞州奧古斯塔 30901）

阿斯巴甜（Aspartame，APM）是一種合成的有機(jī)氨基酸衍生物[1]，是被廣泛使用的低能量和非碳水化合物甜味劑的食品添加劑[2]，其結(jié)構(gòu)見(jiàn)圖1。近年來(lái)，APM與其他添加劑一起使用，如在食物或化妝品中與防腐劑、抗氧化劑產(chǎn)生協(xié)同作用[3]。而APM在低pH和高溫下迅速降解為環(huán)境污染物。在環(huán)境中APM 可降解為氨、酸、醇、醛等有害物，在活體內(nèi)APM 可在胃腸道代謝為苯丙氨酸、天冬氨酸和甲酸3種成分[4]。此外，過(guò)量使用APM 會(huì)導(dǎo)致許多健康問(wèn)題，諸如神經(jīng)紊亂、聽(tīng)力喪失、記憶力減弱，甚至癌癥等疾病[5-7]。因此，APM的分析具有挑戰(zhàn)性和實(shí)用性。

美拉德反應(yīng)，又稱(chēng)非酶褐變反應(yīng)[8-9]，涉及羰基化合物（還原糖類(lèi)）和氨基酸化合物（氨基酸和蛋白質(zhì)類(lèi)）之間的縮合[10]，形成具有濃郁香味的類(lèi)黑精大分子化合物及其相關(guān)的復(fù)雜混合物。美拉德反應(yīng)主要應(yīng)用于食品工業(yè)和香精領(lǐng)域[11]。

圖1 APM的結(jié)構(gòu)圖（L-天門(mén)冬氨酰-L-苯基丙氨酸甲酯）Fig.1 The structure of APM (L-aspartyl-L-phenylalanine methyl ester)

近年研究表明，熒光檢測(cè)因在許多環(huán)境和生物過(guò)程中的敏感性、選擇性、簡(jiǎn)潔性和無(wú)損成像特性而備受關(guān)注[12-15]。此外，共振瑞利散射（resonance rayleigh scattering，RRS）作為一種新穎快捷的分析技術(shù)，已應(yīng)用于許多領(lǐng)域包括生物大分子藥物及其環(huán)境等[16-18]。已有報(bào)道熒光、RRS探針或傳感器[19-20]，可同時(shí)用作熒光和共振瑞利散射的傳感平臺(tái)則很少見(jiàn)，特別是基于熒光和RRS 的同時(shí)變化檢測(cè)APM的傳感平臺(tái)還少見(jiàn)報(bào)道。

本研究利用葡萄糖和L-精氨酸（glucose and L-Arginine，GLA）對(duì)Cu2+的螯合能力，以GLACu2+作為熒光和RRS 傳感平臺(tái)檢測(cè)APM。當(dāng)APM被引入體系時(shí)，熒光形成先猝滅后恢復(fù)的“關(guān)-開(kāi)”模式，而RRS 處于先增強(qiáng)后降低的“開(kāi)-關(guān)”狀態(tài)。另外，熒光恢復(fù)程度和RRS 減弱程度都與APM 濃度呈線性關(guān)系，因此，在后續(xù)的研究中，通過(guò)兩個(gè)線性正相關(guān)方程可以很容易地得到APM的檢測(cè)結(jié)果。這說(shuō)明利用GLA-Cu2+的光譜特征變化作為檢測(cè)APM 的傳感平臺(tái)是有效和可靠的。因此，本研究提出了一種用于檢測(cè)APM 的傳感平臺(tái)GLA-Cu2+。實(shí)驗(yàn)結(jié)果還表明，該平臺(tái)具有良好的光學(xué)性質(zhì)，對(duì)APM 的測(cè)定具有較高的靈敏度和選擇性。傳感平臺(tái)的設(shè)計(jì)運(yùn)用將有利于進(jìn)行實(shí)時(shí)環(huán)境分析和生物分析。

1 實(shí)驗(yàn)

1.1 儀器

采用日立F-4500熒光分光光度計(jì)(日本日立公司)記錄熒光光譜，狹縫為10 nm。采用英國(guó)愛(ài)丁堡儀器有限公司的近紅外熒光光譜儀FS5 記錄了RRS 光譜，狹縫為5 nm。紫外可見(jiàn)UV 2700 分光光度計(jì)（日本島津儀器設(shè)備有限公司）被用于記錄吸收光譜。采用pHS-3C-02（上海三信儀器設(shè)備有限公司，中國(guó)上海）測(cè)定溶液的pH。所有測(cè)量都在正常的實(shí)驗(yàn)環(huán)境條件下進(jìn)行。

1.2 試劑

購(gòu)置的化學(xué)品和試劑都為分析純級(jí)。實(shí)驗(yàn)用水是雙蒸餾水。主要化學(xué)試劑為：D-葡萄糖（C6H12O6·H2O，西隆化工有限公司，中國(guó)廣州），L-精氨酸（C6H14N4O2，阿拉丁實(shí)業(yè)有限公司，中國(guó)上海），三水合硝酸銅（II）[Cu（NO3）2·3H2O，國(guó)藥控股有限公司，中國(guó)上海]，氫氧化鈉（NaOH，西隆化工有限公司，中國(guó)四川），阿斯巴甜（C14H18N2O5，阿拉丁實(shí)業(yè)有限公司，中國(guó)上海)，以及羥乙基哌嗪乙硫磺酸HEPES（C8H18N2O4S，百靈威科技有限公司，中國(guó)北京)。

制備0.01 mol/L Cu2+和0.01 mol/L APM 的儲(chǔ)備液。不同pH 的羥乙基哌嗪乙硫磺酸緩沖液制備：將0.2 mol/L NaOH 與0.4 mol/L HEPES 儲(chǔ)備液按不同比例混合配制不同pH 的HEPES 緩沖液，用pH計(jì)校準(zhǔn)。

1.3 具有光學(xué)活性的水溶性GLA探針的制備

美拉德反應(yīng)產(chǎn)物合成：先將葡萄糖（Glu，0.168 4 g）、L-精氨酸粉末（L-Arg，0.087 2 g）、9.5 mL 超純水加入三頸燒瓶至滿(mǎn)，攪拌1 min。加入0.5 mL 1 mol/L NaOH 溶液再攪拌1 min 形成均勻的溶液。隨后，在95 ℃下加熱攪拌1 h。最終的溶液經(jīng)過(guò)濾后作為傳感平臺(tái)的活性物質(zhì)。該活性溶液的濃度為0.05 mol/L，標(biāo)名為“GLA”，存放在-4 ℃的冰箱中備用。

1.4 熒光和共振瑞利散射實(shí)驗(yàn)

首先，將GLA 溶液稀釋10 倍用于整個(gè)熒光和RRS 實(shí)驗(yàn)。將0.8 mL HEPES 緩沖液（pH=7.4）和0.2 mL 稀釋的GLA 溶液依次加入10 mL 校準(zhǔn)的試管中。隨后，在混合溶液中加入1.0 mL 0.01 mol/L Cu2+溶液并分別加入系列濃度的APM溶液。然后稀釋至刻度、混合、靜置10 min，設(shè)置激發(fā)波長(zhǎng)為334 nm，掃描采集溶液在300～600 nm 波段內(nèi)的熒光光譜。結(jié)果分析以相對(duì)熒光強(qiáng)度ΔF=F1-F0記錄（F0、F1分別為GLA-Cu2+體系中沒(méi)有和有APM時(shí)的熒光強(qiáng)度），當(dāng)λex=λem，記錄體系的RRS 光譜，ΔIRRS=IRRS-I0RRS，IRRS為反應(yīng)產(chǎn)物的散射強(qiáng)度，I0RRS為空白的散射強(qiáng)度。

2 結(jié)果與討論

2.1 GLA的光譜特征

為了進(jìn)一步研究合成的GLA 的光學(xué)性質(zhì)和配體的化學(xué)結(jié)構(gòu)，記錄了GLA 的UV-vis 吸收光譜和熒光光譜，如圖2（a）所示。GLA 的特征吸收峰位于295 nm 處。當(dāng)激發(fā)波長(zhǎng)為334 nm，GLA 的熒光光譜在408 nm 處有發(fā)射峰，所以GLA 的激發(fā)波長(zhǎng)與發(fā)射波長(zhǎng)之間的斯托克位移為74 nm，說(shuō)明可以消除背景干擾。同時(shí)，我們還比較了美拉德反應(yīng)前后的熒光光譜，如圖2（b）所示。在相同的實(shí)驗(yàn)條件下，美拉德反應(yīng)的反應(yīng)物（Glu和L-Arg）的發(fā)射熒光較GLA產(chǎn)物弱，表明亮藍(lán)色熒光來(lái)自GLA。

圖2 GLA的UV-Vis吸收光譜和熒光光譜（a)以及Glu,L-Arg 和GLA的熒光光譜比較（b）（cL-Arg∶cGlu=1∶2，cGLA=1.0 mmol/L）Fig.2 (a)The UV-Vis absorption and fluorescence spectra of GLA; (b)Comparison of fluorescence spectra of Glu, L-Arg and GLA (cL-Arg∶cGlu =1∶2, cGLA=1.0 mmol/L)

2.2 熒光“關(guān)-開(kāi)”模式檢測(cè)APM的響應(yīng)

制備的GLA為水溶性，具有突出的藍(lán)色熒光。由圖3 可知，GLA 的最大熒光發(fā)射波長(zhǎng)為408 nm。在Cu2+存在下，GLA的熒光強(qiáng)度明顯減弱，藍(lán)色熒光消退為無(wú)色。然而，當(dāng)將APM 加入體系中時(shí)，熒光強(qiáng)度呈線性恢復(fù)，再次出現(xiàn)藍(lán)色熒光。因此，GLA-Cu2+可以作為檢測(cè)APM 有效可靠的熒光檢測(cè)平臺(tái)。

圖3 GLA（a），GLA-Cu2+（b），GLA-Cu2+-APM（c）的熒光光譜Fig.3 The fluorescence spectra of GLA (a), GLA-Cu2+(b),and GLA-Cu2+-APM(c)

2.3 RRS“開(kāi)-關(guān)”響應(yīng)檢測(cè)APM

為進(jìn)一步研究GLA- Cu2+與APM 之間的反應(yīng)，記錄了GLA- Cu2+-APM 體系的RRS 光譜。如圖4所示，GLA 的RRS 強(qiáng)度很弱，并且沒(méi)有明顯的RRS特征峰。在GLA溶液中加入Cu2+后，在328 nm處出現(xiàn)新的RRS 特征峰。然而，當(dāng)加入APM 后，RRS 的強(qiáng)度呈線性降低。因此，基于RRS 強(qiáng)度在此不同情況下的簡(jiǎn)單變化，可用Cu2+結(jié)合GLA開(kāi)發(fā)一種用于APM檢測(cè)的有效的RRS傳感平臺(tái)。

圖4 GLA，GLA-Cu2+，GLA-Cu2+-APM的RRS光譜Fig.4 TheRRSspectraofGLA,GLA-Cu2+,andGLA-Cu2+-APM

2.4 條件優(yōu)化

2.4.1 GLA合成條件的優(yōu)化

為了獲得GLA 最高的熒光強(qiáng)度，對(duì)GLA 的一系列合成條件進(jìn)行了研究。首先，通過(guò)改變Glu的量，同時(shí)固定L-Arg 的量，研究了兩種反應(yīng)前體對(duì)GLA 熒光強(qiáng)度的影響。結(jié)果表明，最佳配比為1∶2[圖5（a）]，并選擇該配比制備GLA作為傳感平臺(tái)；其次，溫度對(duì)美拉德反應(yīng)速率有重要影響，圖5（b）表明了溫度對(duì)反應(yīng)速率的影響，95 ℃被選為合成溫度；然后考察合成時(shí)間，如圖5（c）所示，獲得GLA 產(chǎn)物需要反應(yīng)1 h；最后，研究了溶液pH對(duì)熒光強(qiáng)度的影響，從圖5（d）可以看出，在pH 為11.25 時(shí)熒光強(qiáng)度達(dá)到最大，為美拉德反應(yīng)的最佳pH。

圖5 GLA溶液制備的熒光優(yōu)化條件：L-Arg/Glu比例(a)，反應(yīng)溫度(b),反應(yīng)時(shí)間(c), pH值(d)Fig.5 The fluorescence spectra optimization conditions of GLA solution preparation: concentration ratios of L-Arg /Glu(a),temperature(b), and reaction time(c), and pH values(d)

2.4.2 GLA-Cu2+檢測(cè)平臺(tái)分析APM的最佳條件

選擇緩沖溶液是最主要也是最基本的步驟。本實(shí)驗(yàn)研究了pH 和濃度相同的6 種緩沖溶液（磷酸二氫鈉-檸檬酸、PBS，Tris-HCl，HEPES，BR，NaH2PO4-Na2HPO4）對(duì)體系的影響。結(jié)果表明，HEPES 的用量很小且反應(yīng)明顯，說(shuō)明HEPES 緩沖溶液比其他緩沖液好。如圖6（a）所示，兩種體系在pH 6.8～8.2條件下均穩(wěn)定。此外，pH 7.4接近生理pH，表明此檢測(cè)平臺(tái)具有潛在的活體生物應(yīng)用優(yōu)勢(shì)。因此，選擇pH 為7.4 的HEPES 緩沖溶液作為最佳反應(yīng)酸度條件。最后研究了緩沖容量的影響，實(shí)驗(yàn)結(jié)果表明選pH 7.4 的HEPES 緩沖液作為反應(yīng)介質(zhì)，取0.8 mL 為宜。本實(shí)驗(yàn)還討論了反應(yīng)時(shí)間對(duì)GLA-Cu2+-APM體系熒光強(qiáng)度的影響，如圖6（b）所示，室溫下，該體系反應(yīng)很快，10 min即可完成。

2.5 檢測(cè)傳感平臺(tái)的選擇性

為研究檢測(cè)傳感平臺(tái)的選擇性，在相同的實(shí)驗(yàn)條件下，研究了GLA 在鈰離子（Ce3+），鉻離子（Cr3+），鐵離子（Fe3+），鋁離子（Al3+），銅離子（Cu2+），鋅離子（Zn2+），鋇離子（Ba2+），鎳離子（Ni2+），鎂離子（Mg2+），錳離子（Mn2+），鈷離子（Co2+），鈣離子（Ca2+），鎘離子（Cd2+），鉛離子（Pb2+），鈀離子（Pd2+）15種常見(jiàn)金屬離子存在下的熒光光譜。同時(shí)，在APM 存在的情況下，對(duì)含有上述所有金屬離子的GLA進(jìn)行了選擇性實(shí)驗(yàn)。如圖7 所示，有3種金屬離子（Fe3+，Cu2+，Pd2+）在408 nm 處對(duì)GLA 有明顯的響應(yīng)。然而，只有在GLA-Cu2+體系中加入APM 后，才能最大程度恢復(fù)其猝滅熒光，因此，GLA-Cu2+作為檢測(cè)傳感平臺(tái)分析APM是有效可靠的。

圖6 緩沖溶液的pH分別對(duì)GLA-Cu2+和GLA-Cu2+-APM體系的影響（a）；優(yōu)化反應(yīng)時(shí)間對(duì)GLA-Cu2+-APM體系的相對(duì)熒光強(qiáng)度的影響（b）（cGLA=1.0 mmol/L，c Cu2+=1.0 mmol/L，cAPM=1.0 mmol/L）Fig.6 (a) Effect of pH value of buffer solution on the fluorescence intensity of GLA-Cu2+ and GLA- Cu2+-APM system；(b) effect of optimization reaction time on the relative fluorescence intensity in GLA-Cu2+-APM system (cGLA=1.0 mmol/L,cCu2+=1.0 mmol/L, cAPM=1.0 mmol/L)

圖7 0.5 mmol/L 的金屬離子對(duì)GLA和GLA-APM的熒光相對(duì)強(qiáng)度的選擇性影響Fig.7 The relative fluorescence intensities of GLA and GLAAPM in the presence of metal cations (Ce3+，Cr3+，F(xiàn)e3+，Al3+，Cu2+，Zn2+，Ba2+，Ni2+，Mg2+，Mn2+，Co2+，Ca2+，Cd2+，Pb2+，Pd2+)with the concentrations of 0.5 mmol/L

2.6 共存物質(zhì)的影響

實(shí)驗(yàn)研究了氨基酸、糖、金屬離子等共存物質(zhì)對(duì)分析測(cè)定APM 的影響，結(jié)果見(jiàn)表1。其中，Pd2+和Fe3+對(duì)實(shí)驗(yàn)結(jié)果有較大的干擾，但可以在樣品預(yù)處理時(shí)通過(guò)添加EDTA消除干擾，其余的共存物質(zhì)包括氨基酸、蛋白質(zhì)、單糖、低聚糖、多糖以及其他金屬離子和無(wú)機(jī)酸自由基對(duì)測(cè)定結(jié)果無(wú)干擾，因此，該平臺(tái)有很強(qiáng)的選擇性和抗干擾能力。

表1 共存物質(zhì)對(duì)分析測(cè)定APM的影響（cAPM=10.0 mmol/L）Tab.1 Effects of coexistence substances on the analysis and determination of APM (cAPM=10.0 mmol/L)

2.7 體系的反應(yīng)機(jī)制

研究涉及化合物結(jié)構(gòu)和光譜信號(hào)的關(guān)系，對(duì)該傳感器的機(jī)制進(jìn)行了探討和推測(cè)。通過(guò)FT-IR研究了GLA，Glu和L-Arg的結(jié)構(gòu)，如圖8（a）所示，在GLA 的紅外光譜中，1 612 cm-1處的峰為C—N鍵，因?yàn)镚lu 的羰基基團(tuán)與L-Arg 的氨基發(fā)生反應(yīng)。1 406 cm-1和1 344 cm-1左右的峰表明存在羰基和羥基。如圖3 中曲線（b）所示，在最優(yōu)條件下，隨著Cu2+的加入，GLA 的熒光強(qiáng)度顯著淬滅，顏色也逐漸變?yōu)榈G色。但在圖4中，將Cu2+加入GLA 溶液中時(shí)，RRS 強(qiáng)度顯著增強(qiáng)，且在328 nm處出現(xiàn)新的RRS 特征峰。熒光強(qiáng)度的降低而RRS強(qiáng)度的增強(qiáng)可能是由于GLA-Cu2+復(fù)合物的形成，因此，熒光和RRS 光譜的變化可能是Cu2+與GLA中的羰基基團(tuán)和羥基配位的結(jié)果[19-20]。為進(jìn)一步驗(yàn)證，研究了GLA-Cu2+體系的傅里葉紅外光譜（FT-IR）。如圖8（b）所示，由于Cu2+與GLA發(fā)生反應(yīng)，GLA在1 406 cm-1和1 344 cm-1處的原峰消失，在1 383 cm-1處出現(xiàn)新峰。

然而，當(dāng)將APM 加入上述體系后，熒光強(qiáng)度隨即恢復(fù)，發(fā)出顏色較淺的光，得到改善[圖3 中曲線（c）]。熒光強(qiáng)度的恢復(fù)可能是由于APM 與Cu2+之間的結(jié)合作用，其結(jié)合力甚至強(qiáng)于Cu2+與GLA 之間的結(jié)合力[1]。另一方面，在GLA- Cu2+體系中加入APM 后，RRS 強(qiáng)度降低，如圖4 所示。眾所周知，RRS 的光譜特征和散射強(qiáng)度受分子大小、形狀、構(gòu)象和界面性質(zhì)的強(qiáng)烈影響，這為研究生物分子間的相互作用提供了有利的新信息[21-22]。因此，RRS 強(qiáng)度的增加可能是加入Cu2+時(shí)，GLA- Cu2+的分子體積增大所致。盡管如此，因?yàn)锳PM 的體積小于GLA，當(dāng)APM 在溶液中取代GLA 時(shí)，新的金屬絡(luò)合物APM- Cu2+的尺寸減小，體系的RRS強(qiáng)度最終減弱。

圖8 單獨(dú)的GLA, Glu ,L-Arg的傅里葉紅外光譜（a）以及體系GLA- Cu2+和GLA- Cu2+-APM的傅里葉紅外光譜（b）Fig.8 The Fourier transform infrared(FT-IR)spectrum of(a)single GLA,Glu,L-Arg and(b)the systems of GLA-Cu2+,GLA-Cu2+-APM

GLA在沒(méi)有和有Cu2+存在時(shí)的紫外可見(jiàn)吸收光譜如圖9 所示，根據(jù)UV-vis 吸收光譜可知，GLA在295 nm 處有一個(gè)吸收峰，但加入Cu2+后，在755 nm 處出現(xiàn)新的吸收峰且GLA 的初始吸收峰消失，說(shuō)明GLA與Cu2+發(fā)生了反應(yīng)，形成了新的復(fù)合物。但加入APM 后，由于GLA 與Cu2+的結(jié)合力較弱，APM與Cu2+的結(jié)合力相對(duì)較強(qiáng)，吸收峰發(fā)生藍(lán)移，特征峰重新出現(xiàn)在655 nm處。因此，GLA-Cu2+體系的熒光恢復(fù)也是Cu2+與APM 相互作用的結(jié)果。APM中含有羧基和氨基，可以通過(guò)螯合作用與Cu2+發(fā)生反應(yīng)，因此，其熒光恢復(fù)和RRS 降低可能是GLA與APM對(duì)Cu2+的競(jìng)爭(zhēng)性置換所致。

圖9 GLA，GLA-Cu2+，GLA-Cu2+-APM以及Cu2+和APM的UV-Vis吸收光譜Fig.9 UV-Vis absorption spectra of GLA, GLA-Cu2+, GLACu2+-APM, APM, Cu2+

2.8 標(biāo)準(zhǔn)曲線

在最優(yōu)條件下，不同濃度APM 的GLA-Cu2+體系的熒光光譜如圖10 所示。根據(jù)結(jié)果分析，相對(duì)熒光恢復(fù)強(qiáng)度△F=F1-F0（F0，F(xiàn)1分別為GLA-Cu2+體系中沒(méi)有和有APM 時(shí)的熒光強(qiáng)度）與濃度在0.3～300 μmol/L 范圍的APM 呈線性關(guān)系，如圖10中插圖所示?；?σ/S的標(biāo)準(zhǔn)偏差（其中σ為測(cè)定空白樣品11 次的標(biāo)準(zhǔn)偏差，S為校準(zhǔn)斜率），檢出限為26 nmol/L，相關(guān)系數(shù)為0.999 5。線性回歸方程為ΔF=56.69c+35.93（c為APM的濃度）。

圖10 不同濃度APM的GLA-Cu2+(cGLA=1.0 mmol/L，c Cu2+=0.5 mmol/L）體系的熒光光譜圖Fig.10 Fluorescence sprectra of GLA-Cu2+ (cGLA=1.0 mmol/L，cCu2+=0.5 mmol/L）system with different concentrations of APM

圖11 討論不同濃度APM 的GLA-Cu2+體系的RRS 光譜。與熒光變化相比，隨著APM 濃度的增加，GLA-Cu2+體系在328 nm 處的RRS 強(qiáng)度逐漸降低。APM 濃度在0.4～800 μmol/L 范圍時(shí)，RRS 強(qiáng)度（IRRS）與APM濃度呈線性關(guān)系，檢出限為39 nmol/L。線性回歸方程為IRRS=-0.942c+7.941（其中c為APM濃度），相關(guān)系數(shù)為0.999 3。

3 本法對(duì)水樣中APM的實(shí)際分析

為驗(yàn)證本方法的可靠性和實(shí)用性，進(jìn)行了實(shí)際水樣中APM 檢測(cè)的標(biāo)準(zhǔn)回收試驗(yàn)。自來(lái)水樣品中未檢出APM，為凈化處理水樣，樣品都用濾紙過(guò)濾。在測(cè)試中，每個(gè)樣本被測(cè)試5 次取平均值。如表2 所示，APM 回收率在97.80%～101.67%之間波動(dòng)，相對(duì)標(biāo)準(zhǔn)偏差小于3%。結(jié)果表明，該方法對(duì)自來(lái)水樣品中APM 的檢測(cè)可靠并具有較高的準(zhǔn)確性。

圖11 不同濃度APM的GLA-Cu2+(cGLA=0.1 mmol/L，c Cu2+=0.5 mmol/L）體系的RRS光譜圖Fig.11 The RRS spectra of of GLA-Cu2+ (cGLA=0.1 mmol/L，cCu2+=0.5 mmol/L）system with different concentrations of APM

表2 自來(lái)水樣中APM檢測(cè)的回收實(shí)驗(yàn)Tab.2 Recovery test of APM in tap water samples

4 結(jié)論

綜上所述，利用Glu 和L-Arg 的美拉德反應(yīng)合成了具有良好的光學(xué)活性和水溶性的GLA，并研究了GLA 的初始光學(xué)性質(zhì)。GLA 是發(fā)藍(lán)色熒光的產(chǎn)物與Cu2+螯合而成的復(fù)合物，加入APM 后對(duì)GLA-Cu2+的熒光具有“關(guān)-開(kāi)”響應(yīng)模式和對(duì)RRS的熒光具有“開(kāi)-關(guān)”響應(yīng)模式，為此可作為FL和RRS 法檢測(cè)APM 的傳感平臺(tái)。結(jié)果表明，這種新型的快速檢測(cè)APM 的傳感平臺(tái)是有效可靠的。因此，可以建立FL法和RRS法對(duì)痕量APM進(jìn)行快速靈敏的檢測(cè)。將該GLA-Cu2+傳感平臺(tái)應(yīng)用于實(shí)際水樣中APM的檢測(cè)，取得了滿(mǎn)意的效果。推測(cè)GLA-Cu2+可以通過(guò)美拉德反應(yīng)作為低成本、光學(xué)性能優(yōu)異的傳感平臺(tái)，擴(kuò)展用于各種其他手性物質(zhì)的檢測(cè)。

Aspartame (APM) is a synthetic organic amino acid derivative[1].It is widely used as a kind of food additive with low-energy artificial and non-carbohydrates sweetener[2].Recently, APM is even mixed with other kinds of additives like preservatives, antioxidants to produce synergistic effects in food or cosmetics[3].However, APM rapidly degrades into environmental contaminants at low pH and high temperatures.In vivo, APM can be metabolized in the gastrointestinal tract into three components: phenylalanine,aspartic acid and methanol[4].Furthermore, the excessive use of aspartame may lead to a number of health problems including neurological disturbances, loss of hearing, memory diseases, and even cancer[5-7].Thus,the analysis of APM is still challenging and practical.

Maillard reaction, known as nonenzymatic browning reaction[8], involves the condensation between the carbonyl compound (reducing sugars) and amino compounds (amino acids and proteins)[10]and forms a complex mixture of the macromolecular compounds and their associated mixtures.Maillard reaction is mostly used in the food industry and essence fields[11].

Recent studies have indicated that fluorescencebased assay has attracted considerable attention inmany environmental and biological processes due to its sensitivity, selectivity, simplicity and nondestructive imaging properties[12-15].Additionally, Resonance Rayleigh scattering (RRS), a novel sensitive analytical technique with convenient performance, has been applied to many fields including biological macromolecules, pharmaceuticals and environment[17-18].In the past, some fluorescent and RRS probes or sensor had been reported[19-20], but the sensing platform that could be simultaneously used for fluorescence and resonance Rayleigh scattering is very few.Especially,the detection of APM based on the fluorescence and RRS changes have never been reported.

Fig.1 The structure of APM (L-aspartyl-L-phenylalanine methyl ester)

In this work, we used GLA-Cu2+as fluorescence and RRS sensing platform to determine APM owing to the chelating ability of GLA toward Cu2+.When introducing APM into the system, the fluorescence was quenched then restored to form the turn-off-on switch, while the RRS was able to be enhanced then reduced in a turn-on-off mode.T extents of both FL recovery and RRS reduction were linearly related to the concentration of APM in a wide range.Therefore, the detection of APM could be easily achieved through the linear relationship in an effective and reliable way.Thus, this paper proposes a sensing platform of GLA-Cu2+for APM detection, which shows excellent optical performance with high sensitivity and selectivity.The design and synthesis of the sensing platform would be beneficial for real-time analysis of environmental and biological problems.

1 Experimentation

1.1 Apparatus

A Hitachi F-4500 fluoro-spectrophotometer (Hitachi Company, Japan) with slits (EX/EM) of 10.0/10.0 nm was used to record the fluorescence spectra.A FS5 near infrared spectrofluorometer (Edinburgh Instruments Ltd, UK) with slits (EX/EM) of 5.0/5.0 nm was used to record resonance Rayleigh scattering spectra.A UV-2700 220V CH spectrophotometer(Shimadza Corporation, Kyoto, Japan）was applied to record the absorption spectra.A pHS-3C-02 meter(Shanghai San-Xin Instrumentation, Shanghai, China)was used to adjust the pH values of the aqueous solutions.All measures were carried out under the normal environmental condition.

1.2 Reagents

All chemicals and reagents of analytic grade were purchased without no additional purification.The doubly distilled water was used throughout the experiments.The main chemical reagents (and their sources) were: D-Glucose (C6H12O6?H2O, Xilong Chemical Co., Guangzhou, China), L-Arginine (C6H14N4O2,Aladdin Industrial Co., Shanghai, China), Copper(II)nitrate trihydrate (Cu(NO3)2?3H2O, Sinopharm Chemical Reagent Co., Shanghai, China), Sodium hydroxide(NaOH, Xilong Chemical Co., Sichuan, China), Aspartame (C14H18N2O5, Aladdin Industrial Co., Shanghai,China), and 2-[4-(2-Hydroxyethyl)-1- piperazine]ethane sulfonic acid HEPES (C8H18N2O4S, Lark technology Co., Beijing, China).

A stock solution of 0.01 mol/L Cu2+and a stock solution of 0.01 mol/L APM were prepared, and the HEPES buffer solutions with different pH values were prepared by mixing 0.2 mol/L NaOH with 0.4 mol/L HEPES stock solution in different proportions, and calibrated by pH meter.

1.3 Preparation of water-soluble GLA with optical activity

The Maillard reaction products were synthesized as follows: all glasswares and magnetic stirrer bars were thoroughly cleaned in aqua regia (HNO3/HCl=1:3,V/V), rinsed thoroughly in water, and then overdried prior to use.Firstly, glucose (Glu, 0.168 4 g)and L-Arginine powders (L-Arg, 0.087 2 g) were added to a three-necked flask filled with 9.5 mL ultra pure water under stirring for 1 min.Then 0.5 mL of 1 mol/L NaOH was added to the above solution(pH 11.25) and stirred for another 1 min to form a homogeneous solution.Subsequently, the mixtures were heated at 95 ℃with stirring for 1 h.The final solution was filtered before used as active substances of the sensing platform.The as-prepared active solution with concentration of 0.05 mol/L, labeled as“GLA”,was finally stored in the refrigerator (0～4 ℃) for the future reaction.

1.4 Fluorescence and Resonance Rayleigh scattering (RRS) assays

Firstly, the GLA solution was diluted 10 times for the entire fluorescence and RRS assays.0.8 mL of HEPES buffer solution (pH=7.4), and 0.2 mL freshly diluted GLA solution were added into 10 mL calibrated test tube in succession.Therewith, 1.0 mL 0.01 mol/L Cu2+solution and a series of APM solution at different concentrations were added into the mixture solution.Then the above solution was diluted, mixed and incubated for 10 min.The fluorescence spectra of the solutions were collected at the emission wavelengthλemfrom 300 nm to 600 nm by setting excitation wavelengthλexat 334 nm.The emission signals were collected for each addition, and the relative intensity of fluorescence recovery was recorded, i.e.ΔF=F1-F0, whereF0andF1were the fluorescence intensity of GLA-Cu2+system in the absence or presence of APM, respectively.The RRS spectra of the system were recorded using synchronous scanning atλex=λem.The scattering intensities,IRRSfor the reaction product andI0RRSfor the reagent blank, were measured at their maximum wavelengths:ΔIRRS=IRRS-I0RRS.

2 Results and discussion

2.1 The spectral characteristics of GLA

In order to further investigate the optical properties and ligand chemical structure of the synthesized GLA, the UV-vis absorption spectrum and fluorescence spectrum of GLA were recorded and shown in Fig.2 (a).The characteristic absorption peak of the prepared GLA was located at 295 nm.The fluorescence spectrum of GLA showed an emission peak at 408 nm under the excitation wavelength of 334 nm,so the stokes shift between the excitation and emission wavelengths of maximum fluorescence intensities of GLA was 74 nm which allows to eliminate the background interference.At the same time, we also compared the fluorescence spectrum before and after the Maillard reaction, as shown in Fig.2 (b).The results demonstrated that the emitting fluorescence of the reactants of Maillard reaction (Glu and L-Arg)was rather weak compared to the products labeld as GLA under the same test addition, indicating the bright blue fluorescence mainly from GLA.

Fig.2 (a)The UV-Vis absorption and fluorescence spectra of GLA; (b)Comparison of fluorescence spectra of Glu, L-Arg and GLA (cL-Arg∶cGlu =1∶2, cGLA=1.0 mmol/L)

2.2 Turn-off-on response of fluorescence for APM detection

The prepared GLA product is water-soluble and has highlight blue-emitting efficiency.Fig.3 showed the maximum fluorescence emission wavelength of GLA at 408 nm.The fluorescence intensity of GLA was greatly quenched in the presence of Cu2+with the blue fluorescence fading into colorless.However, assoon as APM was added into the system, the fluorescence intensity could be recovered in a linear fashion with the added APM concentration, and the blueemitting light appeared again nearly instantly.So,GLA-Cu2+could act as an effective sensing platform of turn-on fluorescence for the determination of APM.

Fig.3 The fluorescence spectra of GLA (a), GLA-Cu2+ (b),and GLA- Cu2+ -APM (c)

2.3 “Turn-on-off”response of RRS for APM detection

Fig.4 TheRRSspectraofGLA,GLA-Cu2+,andGLA-Cu2+-APM

In order to further investigate the interaction between GLA-Cu2+and APM, the RRS spectra in the GLA-Cu2+-APM system were recorded.As shown in Fig.4 , the RRS intensities of GLA was very low without no any obvious characteristic peak.After Cu2+was added to GLA solution, a new characteristic RRS peak appeared at 328 nm.Nevertheless, the RRS intensity could linearly decrease with the addition of APM.Therefore,based on the linear intensity change of RRS,an effective RRS sensing platform was developed from the combination of Cu2+-GLA to determine APM.

2.4 Optimum conditions for the reactions

2.4.1 The optimal synthesis condition of GLA

To achieve the highest fluorescence intensity of GLA, a series of synthesis conditions were investigated.Firstly, the effect of two reaction precursors on the fluorescence intensity was investigated via changing the amount of Glu while fixing the amount of L-Arg.The best mixing ratio was found to be 1∶2 [Fig.5 (a)]and selected to prepare GLA for the sensing platform.Secondly, temperature plays an important role in the reaction rate of Maillard reaction [Fig.5 (b)], and 95 ℃was selected as synthesis temperature.Thirdly, the synthesis time was investigated [Fig.5 (c)], and fiund that GLA product could be synthesized for 1 h.Finally,the effect of pH values of the mixture on the fluorescence intensity has been studied.Fig.5 (d) illustrated that the fluorescence intensity reached to a maximum at pH 11.25, which was selected as the optimum pH value for Maillard reaction.

2.4.2 The optimum conditions of GLA-Cu2+sensing platform for the determination of APM

It is the most fundamental and essential step to select buffer solution.In this work, the influence of six buffer solutions (NaH2PO4-citric acid, PBS, Tris-HCl, HEPES, BR, and NaH2PO4-Na2HPO4) on the system was investigated at the same pH value and concentration.The effect of HEPES was obvious even for a small amount of it, indicating that the HEPES buffer solution was better than the others.The influence of different pH values was also investigated[Fig.6 (a) ], and the two systems were found to be stable at pH values of 6.8～8.2.The pH range covers the physiological pH of 7.4, and suggests a potential advantage for this sensing platform to be applied to living body.Therefore, The HEPES buffer solution of pH 7.4 was chosen as the optimal reaction aciditycondition.Subsequently, the effect of buffer volume was also studied, and the appropriate amount was chosen as 0.8 mL.

Fig.5 The fluorescence spectra optimization conditions of GLA solution preparation: concentration ratios of L-Arg /Glu(a),temperature(b), and reaction time(c), and pH values(d)

The influence of reaction time on the fluorescence intensity of GLA-Cu2+-APM system has been discussed [Fig.6 (b) ].The results demonstrated that the reaction was very quick and could be accomplished in 10 min at room temperature.

2.5 Selectivity test of the sensing platform

To investigate the selectivity of the sensing platform, the fluorescence spectrum of GLA was studied under the same experimental conditions in the pres-ence of 15 kinds of common metal ions， such as Ce3+, Cr3+, Fe3+, Al3+, Cu2+, Zn2+, Ba2+, Ni2+, Mg2+, Mn2+,Co2+, Ca2+, Cd2+, Pb2+, Pd2+.Meanwhile, the selectivity experiments were also carried out for GLA containing all above metal ions and in the presence of APM.As shown in Fig.7 , three kinds of metal ions (Fe3+, Cu2+,Pd2+) could obviously respond to GLA at 408 nm.However, only the GLA-Cu2+system could mostly recover its quenched fluorescence after the addition of APM.Thus, GLA-Cu2+system, working as a sensing platform for the determination of APM, was effective and reliable.

Fig.6 (a) Effect of pH value of buffer solution on the fluorescence intensity of GLA-Cu2+ and GLA- Cu2+-APM system；(b) effect of optimization reaction time on the relative fluorescence intensity in GLA-Cu2+-APM system (cGLA=1.0 mmol/L,cCu2+=1.0 mmol/L, cAPM=1.0 mmol/L)

2.6 The effect of coexisting substances

The effects of the coexistence with substances such as amino acids, sugars, and metal ions on the determination of APM were investigated here.All the results were listed in Tab.1 .Of all the analysis, Pd2+and Fe3+had larger disturbance on the results but could be easily removed by adding EDTA in sample pretreatment.The rest of coexisting substances had no interference for the determination purpose, including amino acids, proteins, simple sugars, oligosaccharides, polysaccharides, as well as other metal ions and inorganic acid radicals.So the platform has strong selectivity and anti-interference ability.

Fig.7 The relative fluorescence intensities of GLA and GLA-APM in the presence of metal cations (Ce3+，Cr3+，F(xiàn)e3+，Al3+，Cu2+，Zn2+，Ba2+，Ni2+，Mg2+，Mn2+，Co2+，Ca2+，Cd2+，Pb2+，Pd2+) with the concentrations of 0.5 mmol/L

Tab.1 Effects of coexistence substances on the analysis and determination of APM (cAPM=10.0 mmol/L)

2.7 Mechanism of the reaction system

The working mechanism of this sensor was discussed and speculated as follows, based on the structure of all involved compounds and spectrum signals.The structures of GLA, Glu and L-Arg were studied through FT-IR as shown in Fig.8 (a).In the IR spectra of GLA, the peak at about 1 612 cm-1could be attributed to the C—N bond due to the reaction be-tween carbanyl group of Glu and amino group of LArg.The peaks at about 1 406 and 1 344 cm-1could indicate the existence of carbanyl group and hydroxyl group.As shown in Fig.3 ,under the optimal conditions,the fluorescence intensity of the GLA was quenched obviously with the addition of Cu2+[curve (b) ], and the color also changed to pale green gradually.However, in Fig.4 , the RRS intensity increased dramatically with a new characteristic RRS peak appearing at 328 nm when adding Cu2+into GLA solution[curve (b) ]The decrease of fluorescence intensity and the increase of RRS intensity may be due to the formation of a GLA-Cu2+complex.Hence, the change of fluorescence and RRS spectrum may result from the coordination of Cu2+to the carbanyl group and hydroxyl group on GLA[19-20].In order to prove our speculation, we further studied the FT-IR spectra of GLA-Cu2+system.As demonstrated in Fig.8 (b), the original peaks of GLA at 1 406 and 1 344 cm-1disappeared and a new peak at about 1 383 cm-1occurred because of the reaction between Cu2+and the GLA.

However, as soon as APM was added into the above system, the fluorescence intensity recovered instantly and the emission light improved with a more shallow color compared to the original GLA[Fig.3 (c)].The recuperation of fluorescence intensity may be caused by the coordinative effect between APM and Cu2+which was even stronger than that between Cu2+and GLA[1].On the other hand, it was found that the RRS intensity decreased after APM was added into the GLA-Cu2+system, as shown in Fig.4 .As we all know, the RRS spectral characteristics and RRS scattering intensity are strongly influenced by the molecular size, shape, conformation, and interfacial properties, which provide favorable new information for the study of the interaction of biological molecules.[21-22]So, the increase of RRS intensity may be due to the increase of the molecular scattering volume of GLACu2+when Cu2+was added.Nonetheless, because the volume of APM is smaller than GLA, the size of the new metal complex (Cu2+- APM) decreased when APM replaced GLA in the solution., Therefore, the RRS intensity of the system eventually quenched.

Fig.8 The Fourier Transform Infrared (FT-IR) spectrum of(a) single GLA, Glu ,L-Arg and (b) the systems of GLA-Cu2+,GLA-Cu2+-APM

Furthermore, the UV-vis absorption spectra of GLA in the absence and presence of Cu2+were recorded.As shown in Fig.9 , the absorption peak of GLA was located at 295 nm.By adding Cu2+, a new peak appeared at 755 nm but the initial peak of GLA disappeared, indicating that GLA reacted with Cu2+and formed a new complex.However, when APM was added, a blue shift of the absorption peak occurred and the characteristic peak was relocated at 655 nm because the binding force between GLA and Cu2+was weakand allowed to make a relative strong binding force between APM and Cu2+.Hence, the fluorescence recovery of GLA-Cu2+system was caused by the interaction of Cu2+with APM as well.APM contains carboxyl group and amino group that could react with Cu2+through chelation, so the fluorescencerecovery and RRS decrease could be attributed to the competitive-displacement between GLA and APM for Cu2+.

Fig.9 UV-Vis absorption spectra of GLA, GLA-Cu2+, GLACu2+-APM, APM, Cu2+

2.8 Calibration curve

Fig.10 Fluorescence sprectra of GLA-Cu2+ (cGLA=1.0 mmol/L，cCu2+=0.5 mmol/L）system with different concentrations of APM

Under the optimal conditions, the fluorescence spectra of GLA-Cu2+system with different concentrations of APM are shown in Fig.10 .The relative fluorescence intensity (fluorescence intensity recovery)was defined as ΔF=F1-F0, whereF0andF1were the fluorescence intensity of GLA-Cu2+system in the absence/presence of APM, respectively.ΔFwas found to be proportional to the concentrations of APM in the ranges of 0.3～300 μmol/L as shown in Fig.10 (inset).The limit of detection was 26 nmol/L based on 3σ/S(whereσis the standard deviation of the 11 blank determinations andSis the slope of the calibration).The linear regression equation was ΔF=56.69c+35.93 (wherecthe concentration of APM).The correlation coefficient was 0.999 5.

The RRS spectra of GLA-Cu2+with different concentrations of APM were also discussed in Fig.11 .Compared to the changes of fluorescence, the RRS intensity at 328 nm of GLA-Cu2+system decreased gradually when the concentration of APM increased.As shown in Fig.11 , the RRS intensity (IRRS) was proportional to the concentration of APM in the ranges of 0.4～800 μmol/L and the limit of detection was 39 nmol/L.The linear regression equation wasIRRS=-0.942c+7.941 (wherecthe concentration of APM with a correlation coefficient of 0.999 3.

Fig.11 The RRS spectra of of GLA-Cu2+ (cGLA=0.1 mmol/L，cCu2+=0.5 mmol/L）system with different concentrations of APM

3 Analysis of APM in water sample by this method

The standard tests for detecting APM in real water samples were also investigated in order to justify the reliability and practicality of the proposed meth-od.The samples were taken from tap water, and no APM was detected.In order to purify the water samples, all of them were filtered by a filter paper.In the experiment, each sample was tested five times to obtain the averaged recovery rates.As shown in Tab.2 ,the mean rates of APM were fluctuated from 97.80%to 101.67%, and the relative standard deviation was less than 3%.The results indicated that the proposed method had a good reliability and accuracy for APM detection in tap water samples.

Tab.2 Recovery test of APM in tap water samples

4 Conclusion

In summary, the GLA with optical activity and water solubility was synthesized by the Maillard reaction of Glu and L-Arg with simple experimental procedures.The basic optical properties of GLA have been studied.GLA-Cu2+complex was formed from the chelation of the highlight blue-emitting fluorescent product with Cu2+and selected as a sensing platform of fluorescence (FL) or resonance Rayleigh scattering (RRS) for the determination of aspartame(APM).The fluorescence turn-off-on response and RRS turn-on-off response of GLA-Cu2+were investigated.Based on the characteristics of the FL and RRS spectra.a novel Sensing Platform for Fast Detection of Aspartame was proposed, which is effective and reliable.This GLA-Cu2+sensing platform has been applied to the detection of APM in real water samples with satisfactory results.Therefore, GLACu2+could serve as the sensing platform with lowcost and excellent optical performance through Maillard reaction to detect other chiral substances for various purposes.