劉長(zhǎng)輝,劉 波,齊風(fēng)佩,王海洋
(湖南城市學(xué)院 材料與化學(xué)工程學(xué)院 黑茶金花湖南省重點(diǎn)實(shí)驗(yàn)室,湖南 益陽(yáng) 413000)
含羥基苯并吲哚的增強(qiáng)型熒光探針測(cè)定過氧化氫
劉長(zhǎng)輝,劉 波,齊風(fēng)佩,王海洋
(湖南城市學(xué)院 材料與化學(xué)工程學(xué)院 黑茶金花湖南省重點(diǎn)實(shí)驗(yàn)室,湖南 益陽(yáng) 413000)
合成了含羥基苯并吲哚并研究其對(duì)過氧化氫的熒光響應(yīng),考察了溶劑種類及pH值等因素對(duì)檢測(cè)體系的影響﹒在最佳實(shí)驗(yàn)條件下,過氧化氫將引發(fā)580 nm處熒光信號(hào)藍(lán)移至560 nm處,且其熒光強(qiáng)度隨過氧化氫濃度的增加而增強(qiáng),線性范圍為0.1~1.2 mmol/L,檢出限為72 mol/L,探針響應(yīng)不受其它干擾物的影響﹒該方法可用于消毒液及自來水樣品中過氧化氫的準(zhǔn)確測(cè)定﹒
過氧化氫;熒光探針;熒光增強(qiáng);苯并吲哚
過氧化氫(H2O2)是一種強(qiáng)氧化劑,廣泛用于醫(yī)用消毒殺菌、農(nóng)業(yè)殺蟲抗菌、工業(yè)漂白發(fā)色和藥物合成等領(lǐng)域,又因其具有漂白、防腐和除臭的效果,常被用作食品添加劑﹒然而,過量的H2O2有致癌性,嚴(yán)重危害人們的身體健康﹒因此,H2O2的高靈敏檢測(cè)在醫(yī)藥、化工及環(huán)境分析等領(lǐng)域受到研究者的密切關(guān)注﹒目前,H2O2的檢測(cè)方法主要有電化學(xué)法[1-3]、色譜法[4]、化學(xué)發(fā)光法[5]、生物發(fā)光法[6]、分光光度法[7-9]和熒光光度法[10-18]等﹒相比上述方法,熒光法具有前處理簡(jiǎn)便、靈敏度高、信號(hào)可調(diào)、快速及操作簡(jiǎn)單等優(yōu)點(diǎn)而備受關(guān)注[19]﹒本文利用 H2O2易將酚羥基氧化為苯醌的反應(yīng)機(jī)制[20-21],設(shè)計(jì)合成了含羥基苯并吲哚,構(gòu)建了一種特異性測(cè)定痕量H2O2的方法,并應(yīng)用于消毒液及自來水樣品中H2O2含量的測(cè)定﹒
U-3010型紫外-可見分光光度計(jì)(日本島津公司);F-4600型熒光光度計(jì)(日本島津公司);Varian INOVA 400核磁共振儀(Varian公司);HP1100 LC/MSD質(zhì)譜儀(Agilent公司)﹒
1,2,2-三甲基苯并吲哚,碘甲烷,3,4-二羥基苯甲醛均購(gòu)自北京百靈威科技有限公司﹒所用試劑均為分析純;實(shí)驗(yàn)用水為超純水系統(tǒng)(Barnstead/Thermolyne Corp.,Dubuque,IA)制備的超純水(18.2 MΩ·cm);溶劑購(gòu)自上海國(guó)藥試劑有限公司,使用前均通過標(biāo)準(zhǔn)方法進(jìn)行純化和干燥﹒樣品均購(gòu)自湖南省益陽(yáng)市主要超市等﹒
含羥基苯并吲哚的合成路線如圖1所示﹒
圖1 含羥基苯并吲哚的合成路線圖
[22]的方法合成:將 1.36 g(6.5 mmol)1,2,2-三甲基苯并吲哚溶于20 mL甲苯中,加入0.8 mL(13 mmol)碘甲烷,混勻回流10 h﹒反應(yīng)完畢,冷卻,抽濾,乙醚洗滌,旋干得白色固體1,2,3,3,-四甲基苯并吲哚,產(chǎn)率51.28%,未經(jīng)純化直接用作后續(xù)反應(yīng)﹒
將176 mg(0.5 mmol)1,2,3,3,-四甲基苯并吲哚與165.6 mg(1.2 mmol)3,4-二羥基苯甲醛溶于15 mL甲苯中,在氮?dú)獗Wo(hù)下回流8 h﹒反應(yīng)完畢,冷卻,加入30 mL去離子水分液,水層用乙酸乙酯提取(50 mL×3)三次,有機(jī)層用飽和食鹽水洗滌(50 mL×2)兩次,合并有機(jī)相,無水硫酸鎂干燥,旋干,硅膠柱層析后得深褐色固體含羥基苯并吲哚,產(chǎn)率17.02%﹒1H NMR(400 MHz, DMSO-d6, δ,ppm): 8.55(d, J=16.2, 2H), 8.40 (d, J=8.1, 2H), 8.32(d, J=8.4, 1H), 8.29(d, J=8.9, 1H), 8.25(d, J=8.1,1H), 7.90(d, J=7.9, 2H), 7.76(m, 2H), 7.66(m, 1H),5.38(s, 2H), 4.02(s, 3H), 2.11(s, 6H).13C NMR (100 MHz, CDCl3, δ, ppm): 186.0, 151.6, 143.2, 140.7,136.8, 134.4, 133.9, 131.9, 130.1, 129.5, 128.9,126.7, 118.5, 57.7, 39.3, 28.1. HRMS-ESI: m/z calcd M+, 344.167 5; found, 344.168 3.
用N, N-二甲基甲酰胺(DMF)溶解DHB并定容得1.0 mmol/L的母液備用﹒將母液用DMF
PBS(10 mmol/L,pH=7.2,體積比1︰4)稀釋至10 mol/L作為探針溶液﹒室溫下,量取1.5 mL探針溶液,加入0.5 mL不同濃度H2O2,測(cè)其紫外-可見吸收光譜;以380 nm激發(fā),測(cè)定其熒光光譜﹒激發(fā)和發(fā)射狹縫均為10 nm并保持不變﹒
測(cè)定方法同1.3,用0.5 mL待測(cè)液替代H2O2標(biāo)準(zhǔn)溶液,利用標(biāo)準(zhǔn)工作曲線計(jì)算求得樣品中H2O2的濃度,每份樣品平行測(cè)定3次﹒
首先考察了探針含羥基苯并吲哚(DHB)與H2O2反應(yīng)前后光物理性質(zhì)的變化情況,結(jié)果如圖2所示﹒
由圖2A可知,DHB的最大吸收波長(zhǎng)位于350 nm處,其與H2O2反應(yīng)30 min后,最大吸收波長(zhǎng)的吸光度增大﹒圖2B說明,探針DHB展現(xiàn)一定強(qiáng)度的熒光信號(hào),其與H2O2作用后的熒光信號(hào)顯著增強(qiáng)﹒可能的原因是,H2O2將酚羥基氧化成苯醌,增大了體系的共軛效應(yīng),改變了體系的推拉電子效應(yīng)﹒
圖2 探針DHB(10 μM)與H2O2(1.0 mM)反應(yīng)前后的吸收光譜(A)和熒光發(fā)射光譜(B)
在工作中,考察了探針 DHB在不同溶劑中的熒光發(fā)射光譜,結(jié)果如圖3所示﹒
由圖3可知,探針DHB在PBS緩沖溶液(10 mmol/L,pH=7.2)中的熒光強(qiáng)度較弱,在乙腈、DMF及乙醇等有機(jī)溶劑中的熒光較強(qiáng),但在DMF-PBS (體積比1︰4)的溶液體系中對(duì)H2O2展現(xiàn)最強(qiáng)的響應(yīng)效果﹒可能的原因是探針 DHB具有較大的共軛體系,其在不同的溶劑體系中展現(xiàn)溶解度的差異﹒因此,在本工作中,檢測(cè)體系均采用 DMF-PBS(體積比 1︰4)的混合溶液作為檢測(cè)體系﹒
圖3 探針DHB(10 μM)在不同溶劑中與H2O2(1.0 mM)反應(yīng)后的熒光發(fā)射光譜
為了確證探針DHB能夠檢測(cè)水體中H2O2,在DMF-PBS (體積比1︰4)緩沖溶液中,我們考察了pH對(duì)檢測(cè)體系的影響,結(jié)果如圖4所示﹒
圖4 探針DHB(10 μM)在不同pH值條件下與H2O2(1.0 mM)反應(yīng)的熒光變化情況
由圖4可知,在pH值4~8的范圍內(nèi),探針DHB保持恒定的熒光信號(hào);加入H2O2并反應(yīng)30 min后,其熒光強(qiáng)度在pH值4~8的范圍內(nèi)的顯著增幅增加﹒然而,在強(qiáng)堿性條件下(pH>8),酚羥基被轉(zhuǎn)化為苯氧負(fù)離子,過氧化氫也容易分解,導(dǎo)致熒光強(qiáng)度的增幅減小,靈敏度偏低﹒結(jié)果表明,DHB能夠在較寬的pH范圍內(nèi)(pH值4~7.4)檢測(cè)H2O2﹒
在DMF-PBS (體積比1︰4)溶液中加入H2O2,考察 H2O2濃度對(duì)檢測(cè)體系的影響,結(jié)果如圖 5所示﹒
由圖5A可知,未加入H2O2時(shí),探針DHB在580 nm處展現(xiàn)較弱的熒光信號(hào);隨著H2O2濃度的增加,560 nm處出現(xiàn)新的熒光信號(hào)且其熒光強(qiáng)度隨H2O2濃度的增加而不斷增強(qiáng);當(dāng)濃度為1.0 mmol/L時(shí),熒光強(qiáng)度增加了3.1倍,且熒光強(qiáng)度在 0.1~1.2 mmol/L的范圍內(nèi)的變化程度與H2O2濃度呈現(xiàn)良好的線性關(guān)系,其回歸方程為y=0.302x+0.071 3,線性相關(guān)系數(shù)R2=0.991 0,最低檢測(cè)限為72 mol/L,如圖5B所示﹒結(jié)果表明,探針DHB可檢測(cè)痕量H2O2﹒
圖5 探針DHB(10 μM)與H2O2作用后熒光發(fā)射光譜(A)及其熒光強(qiáng)度比值((F-F0)/F0)與H2O2濃度的變化曲線(B)
在 DMF-PBS(體積比 1︰4)緩沖溶液中,在pH值為7.2,H2O2濃度為1.0 mmol/L的條件下,我們研究了探針 DHB與可能存在的干擾物作用后的熒光發(fā)射光譜及強(qiáng)度變化,如圖6所示﹒
由圖6可知,探針DHB與水體中可能的干擾物,如 Cl-,Na+,F(xiàn)-,K+,Ca2+,CO32-,F(xiàn)e2+,Mg2+,Zn2+,Ni2+,Cu2+,Pb2+,F(xiàn)e3+,ClO-,等作用后,體系的熒光強(qiáng)度變化可以忽略不計(jì)﹒與其形成鮮明對(duì)比的是,H2O2的加入顯著增強(qiáng)了體系的熒光強(qiáng)度,說明探針DHB對(duì)H2O2展現(xiàn)特異性響應(yīng)能力﹒尤其是,在干擾物共存的條件下,探針DHB與H2O2作用后仍展現(xiàn)很強(qiáng)的熒光信號(hào)﹒結(jié)果表明,探針DHB對(duì) H2O2展現(xiàn)較高的選擇性以及較強(qiáng)的抗干擾能力,為H2O2的特異性定量檢測(cè)提供了保證﹒
圖6 探針DHB(10 μmol/L)與干擾物(1.0 mmol/L)作用后熒光響應(yīng)(A)及干擾物存在下與H2O2作用后熒光響應(yīng)(B)
在 DMF-PBS(體積比 1︰4)緩沖溶液中,在pH值為7.2條件下,將探針DHB用于醫(yī)用雙氧水消毒劑及自來水中H2O2的定量檢測(cè),結(jié)果如表1所示﹒從表1可以看出,探針DHB對(duì)醫(yī)用雙氧水消毒劑中H2O2的加標(biāo)回收率為98.7%~102.2%,對(duì)自來水中H2O2的加標(biāo)回收率為99.7%~103.0%,獲得滿意的測(cè)定結(jié)果﹒因此,探針 DHB可有效測(cè)定實(shí)際樣品中的H2O2﹒
根據(jù)H2O2氧化苯酚為苯醌的機(jī)制,建立了含羥基苯并吲哚的增強(qiáng)型熒光法檢測(cè) H2O2的新體系﹒在DMF-PBS(體積比1︰4)緩沖溶液及pH值為7.2的條件下,H2O2濃度在0.1~1.2 mmol/L范圍內(nèi)與檢測(cè)體系的熒光強(qiáng)度變化呈線性關(guān)系,檢測(cè)限為72 mol/L﹒該方法操作簡(jiǎn)單、選擇性高、抗干擾能力強(qiáng),可應(yīng)用于消毒液及自來水等樣品中痕量 H2O2的測(cè)定,加標(biāo)回收率分別為98.7%~102.2%和 99.7%~103.0%,獲得滿意的測(cè)定結(jié)果﹒
表1 實(shí)際樣品中測(cè)定H2O2
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(責(zé)任編校:陳健瓊)
An Enhanced Fluorescent Probe for the Determination of Hydrogen Peroxide Using Benzoindoles Containing Hydroxy
LIU Chang-hui,LIU Bo,QI Feng-pei,WANG Hai-yang
(College of Materials and Chemical Engineering, Hunan Provincial Key Lab of Dark Tea and Jin-hua, Hunan City University, Yiyang, Hunan 413000, China)
Benzoindoles containing hydroxyl (DHB) was synthesized and applied for the fluorescence response to hydrogen peroxide (H2O2), and the effects of solvents and pH values were studied on the detection system. Under the optimum conditions, the increased concentration of H2O2caused the fluorescence signal of DHB shifted from 580 nm to 560 nm, and that at 560 nm enhanced gradually in the linear range of 0.1 mM to 1.2 mM with detection limit of 0.72 M. This method can be used for the determi nation of trace concentration of H2O2in disinfectant and tapwater samples with satisfactory results and good selectivity over other interference.
hydrogen peroxide; fluorescence probe; fluorescence enhancement; benzoindoles
O657
A
10.3969/j.issn.1672-7304.2017.03.0015
1672–7304(2017)03–0065–05
2017-05-08
國(guó)家自然科學(xué)基金項(xiàng)目(21675042);湖南省教育廳科研項(xiàng)目(15B043);黑茶金花湖南省重點(diǎn)實(shí)驗(yàn)室開放基金項(xiàng)目(HC6120)
劉長(zhǎng)輝(1976-),男,湖南衡陽(yáng)人,副教授,博士,主要從事化學(xué)分析與生物傳感研究﹒E-mail: changzi915@126.com.