CN116425694B - 一种特异性识别丁酰胆碱酯酶的荧光探针LAN-bche及其制备方法和应用 - Google Patents
一种特异性识别丁酰胆碱酯酶的荧光探针LAN-bche及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种特异性识别丁酰胆碱酯酶的荧光探针LAN‑bche及其制备方法和应用，属于荧光探针技术领域。本发明提供了一种特异性识别丁酰胆碱酯酶的荧光探针LAN‑bche，基于ICT机理响应BChE，具有稳定性高、选择性好、水溶性好、灵敏度高和检出限低等优良性质，能够通过荧光法和比色法双模式对BChE进行定量检测。本发明所述荧光探针LAN‑bche能够在300倍稀释的人血清中精准检测BChE含量，且细胞毒性低和生物安全性高，因此可将所述探针LAN‑bche应用于细胞成像和活体成像，可将所述荧光探针LAN‑bche开发为与BChE相关疾病的诊断工具。
Description
技术领域
本发明属于荧光探针技术领域，具体涉及一种特异性识别丁酰胆碱酯酶的荧光探针LAN-bche及其制备方法和应用。
背景技术
生活系统中酶水平的异常总是与各种疾病有关，这使得酶成为各种疾病的典型生物标志物。丁酰胆碱酯酶(BChE；EC3.1.1.8)作为一种丝氨酸水解酶，也被称为血清胆碱酯酶，是胆碱酯酶的重要成员之一，可以水解一些酯类物质，如胆碱[1]，在生物代谢和调节过程中起重要作用[2]。丁酰胆碱酯酶由人肝合成，广泛分布于肝脏、大脑、血液等许多组织中[3]，因此，丁酰胆碱酯酶的活性可以在很大程度上反映肝脏的状况及其产生蛋白质的能力[4]。近年来，临床诊断的肝病越来越受到重视，因为肝脏的一定损伤可能导致丁酰胆碱酯酶生成的减少[5]。丁酰胆碱酯酶同时又是糖尿病的生物标志物[6]。因此，准确检测丁酰胆碱酯酶活性对于在临床诊断中评估肝功能和其相关疾病至关重要。
目前已知的可用于测定丁酰胆碱酯酶含量的方法，如酶联免疫吸附法(ELISA)[7]、拉曼法[8]、电化学分析法[9]和Ellman比色法[10]。但是这些方法均存在设备成本高、操作复杂、灵敏度低或稳定性差而不适用于细胞内和体内检测的问题。
参考文献：
[1]MA J,MA L,CAO L,et al.Point-of-Care Testing ofButyrylcholinesterase Activity through Modulating the Photothermal Effect ofCuprous Oxide Nanoparticles[J].MikrochimActa.2021,188:392.
[2]YANG S H,SUN Q,XIONG H,et al.Discovery of a Butyrylcholinesterase-Specific Probe Via a Structure-Based Design Strategy[J].Chem Commun(Camb).2017,53:3952-3955.
[3]YAO J,YUAN Y,ZHENG F,et al.Unexpected Reaction Pathway forButyrylcholinesterase-Catalyzed Inactivation of"Hunger Hormone"Ghrelin[J].SciRep.2016,6:22322.
[4]XIANG C,XIANG J,YANG X,et al.Ratiometric Imaging ofButyrylcholinesterase Activity in Mice with Nonalcoholic Fatty Liver Using anAie-Based Fluorescent Probe[J].J Mater Chem B.2022,10:4254-4260.
[5]QU Z,YU T,LIU Y,et al.Determination of ButyrylcholinesteraseActivity Based on Thiamine Luminescence Modulated by Mno(2)Nanosheets[J].Talanta.2021,224:121831.
[6]CAO T,ZHENG L,ZHANG L,et al.A Highly ButyrylcholinesteraseSelective Red-Emissive Mitochondria-Targeted Fluorescent Indicator Imaging inLiver Tissue of Mice[J].Sensors and Actuators B:Chemical.2021,330
[7]WANG L,DU D,LU D,et al.Enzyme-Linked Immunosorbent Assay forDetection of Organophosphorylated Butyrylcholinesterase:A Biomarker ofExposure to Organophosphate Agents[J].Anal Chim Acta.2011,693:1-6.
[8]LIRON Z,ZIFMAN A,HELEG-SHABTAI V.Surface-Enhanced Raman ScatteringDetection of Cholinesterase Inhibitors[J].Anal Chim Acta.2011,703:234-238.
[9]TENG Y,JIA X,LI J,et al.Ratiometric Fluorescence Detection ofTyrosinase Activity and Dopamine Using Thiolate-Protected Gold Nanoclusters[J].Anal Chem.2015,87:4897-4902.
[10]DING J,QIN W.Potentiometric Sensing of ButyrylcholinesteraseBased on in Situ Generation and Detection of Substrates[J].Chem Commun(Camb).2009:971-973.
发明内容
本发明的目的在于提供了一种特异性识别丁酰胆碱酯酶的荧光探针LAN-bche及其制备方法和应用，所述荧光探针LAN-bche基于ICT机理响应APN，实现对细胞以及组织内APN的荧光成像，具有稳定性高、选择性好、水溶性好、灵敏度高、检出限低等优势。
本发明提供了一种特异性识别丁酰胆碱酯酶的荧光探针LAN-bche，所述荧光探针LAN-bche的分子结构如式I所示，
本发明还提供了上述荧光探针LAN-bche的制备方法，包括以下步骤：将LAN-OH与环丙甲酰氯混合后进行取代反应，得式I所示的荧光探针LAN-bche。
优选的，所述LAN-OH具有如式II所示的结构；
优选的，冰浴条件下，将LAN-OH、环丙甲酰氯和绝干二氯甲烷混合，在三乙胺作用下反应，反应产物中包括式I所示的荧光探针LAN-bche。
本发明还提供了上述荧光探针LAN-bche的在制备检测丁酰胆碱酯酶的产品中的应用。
本发明还提供了上述荧光探针LAN-bche在制备疾病诊断产品中的应用，所述疾病的诊断生物标志物包括丁酰胆碱酯酶。
优选的，所述疾病包括肝脏疾病和/或糖尿病。
优选的，所述疾病诊断产品为可视化诊断产品。
优选的，所述疾病诊断产品基于荧光法或比色法完成可视化诊断。
优选的，所述疾病诊断产品的诊断样本包括血清。
有益效果：本发明提供了一种特异性识别丁酰胆碱酯酶的荧光探针LAN-bche，基于ICT机理响应BChE，具有稳定性高、选择性好、水溶性好、灵敏度高和检出限低等优良性质，能够通过荧光法和比色法双模式对BChE进行定量检测。
本发明所述荧光探针LAN-bche在人血清中具有良好的加标回收率，能够用于人血清中BChE含量的精准检测。本发明实施例中，基于低细胞毒性和高生物安全性，可将所述荧光探针LAN-bche应用于生物成像领域。在细胞成像中，探针LAN-bche被证明能够成像细胞内源性BChE水平并证实肝癌细胞和APAP诱导肝损伤细胞内源性BChE水平均较正常肝细胞下调，这可能是因为肝细胞的癌变和损伤导致其合成BChE的能力遭到破坏，并且探针LAN-bche能够轻松区分混合细胞中的HepG2细胞和LO2细胞。在活体成像中，探针LAN-bche被成功用于APAP诱导的肝损伤和糖尿病小鼠模型内源BChE的成像，证明BChE水平在APAP诱导的肝损伤小鼠体内下调而在糖尿病小鼠体内上调。因此，探针LAN-bche是一种精准检测和诊断BChE相关疾病的有效手段。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1为荧光探针LAN-bche的完整合成路线图；
图2为荧光探针LAN-bche检测BChE的机理示意图；
图3为溶剂DMSO-d6中化合物LAN-OH的1H-NMR谱；
图4为溶剂DMSO-d6中化合物LAN-OH的13C-NMR谱；
图5为化合物LAN-OH的LC-HRMS谱；
图6为溶剂CDCl3中探针LAN-bche的1H-NMR谱；
图7为溶剂CDCl3中探针LAN-bche的13C-NMR谱；
图8为探针LAN-bche的质谱；
图9为探针LAN-bche的光谱特性结果图；(A)、(B)为探针(10μM)和反应体系(10μM探针与700U/LBChE)的荧光光谱/吸收光谱；(C)为探针(10μM)和反应体系(10μM探针与700U/LBChE)在连续580nm激发光照射下的光稳定性；(D)为探针(10μM)和反应体系(10μM探针与700U/LBChE)在25～42℃温度内的荧光变化情况；(E)为10μM探针与不同浓度BChE反应时间变化情况；(F)为pH对探针(10μM)和反应体系(10μM探针与700U/LBChE)的影响；
图10为探针LAN-bche的分析性能结果图；(A)为探针LAN-bche与不同浓度BChE(0～700U/L)响应的荧光光谱；(B)为反应体系在642nm处的荧光强度与BChE浓度(0～200U/L)之间的线性关系；(C)为探针LAN-bche与不同浓度BChE(0～700U/L)响应的吸收光谱；(D)为反应体系在575nm处的吸光度与BChE浓度(0～200U/L)之间的线性关系；
图11为荧光探针LAN-bche与BChE之间的反应动力学研究结果；(A)为200U/LBChE与不同浓度(1，2，3，4，5，6，7，8，9，10μΜ)探针LAN-bche酶促反应的Michaelis-Menten图；(B)为酶促反应的Lineweaver-bulk图；
图12为在各种分析物存在时(除非另有说明否则均为1mM)探针LAN-bche的荧光强度，图中：1.Blank；2.Mn2+；3.Li+；4.Cu2+；5.Mg2+；6.Ag+；7.Hg2+；8.Ca2+；9.Cd2+；10.Pb2+；11.Ni2+；12.Al3+；13.Zn2+；14.K+；15.Ba2+；16.Fe2+；17.Co2+；18.Na+；19.Fe3+；20.AcO-；21.CN-；22.ClO4-；23.HSO4-；24.SCN-；25.CN-；26.H2PO4-；27.F-；28.Cl-；29.Br-；30.I-；31.Lysine；32.Valine；33.Tryptophan；34.Asparticacid；35.Histidine；36.L-Arginine；37.L-Alanine；38.DL-Homocysteine；39.DL-Phenylalanine；40.L-Cysteine；41.L-Leucine；42.DL-Methionine；43.Glycine；44.L-Glutamic；45.D(+)-Mannose；46.D-Fructose；47.Dithiothreitol；48.L-Ascorbic；49.Chitosan；50.Citrulline；51.5000U/LAlkalinePhosphatase；52.1μMThrombin；53.10mg/LGlucoseOxidase；54.10mg/LGlucoseOxidase；55.10mg/LCarcinoembryonicAntigen；56.10mg/LAlbuminBovineV；57.20mg/LHyaluronidase；58.7000U/LMaltase；59.1000U/LAChE；60.200U/LBChE；
图13为荧光团LAN-OH的质谱；
图14为反应体系的质谱；
图15为探针LAN-bche(10μM)、荧光团LAN-OH(10μM)、反应体系(10μM探针LAN-bche与200U/LBChE)的HPLC分析结果图；
图16为抑制剂Tacrine对探针LAN-bche(10μM)、荧光团LAN-OH(10μM)及反应体系(10μM探针LAN-bche与200U/LBChE)荧光强度的影响图；
图17为探针LAN-bche与BChE的分子对接模拟及探针LAN-bche与荧光团LAN-OH的HOMO/LUMO能级图；
图18为不同浓度探针LAN-bche(0，5，10，20，30，50μM)的细胞毒性；
图19为探针LAN-bche的溶血分析结果图；
图20为探针LAN-bche在细胞内成像随时间的变化规律图；(A)为细胞内荧光强度随时间变化趋势；(B)为对应(A)中细胞内平均荧光强度值；
图21为探针LAN-bche应用于细胞内源性BChE成像结果图；(A)为LO2细胞和HepG2细胞内源BChE荧光成像；(B)为对应(A)中细胞内平均荧光强度值；
图22为探针LAN-bche对APAP诱导的肝损伤细胞内BChE水平的监测图；(A)为对APAP诱导肝损伤细胞内BChE水平波动的监测；(B)为对应(A)中细胞内平均荧光强度值；
图23为探针LAN-bche对LO2和HepG2的混合细胞的区分结果图；
图24为(A)对照组、(B)肝损伤组、(C)糖尿病组以及(D)糖尿病治疗组小鼠尾静脉注射探针LAN-bche(200μM，100μL)后的成像结果图。
具体实施方式
本发明提供了一种特异性识别丁酰胆碱酯酶的荧光探针LAN-bche，所述荧光探针LAN-bche的分子结构如式I所示，
本发明所述荧光探针LAN-bche可通过荧光法和比色法有效地检测BChE，在荧光光谱中，在580nm激发光的照射下，加入BChE后探针LAN-bche在642nm处的荧光强度显著增强并伴随着溶液颜色由荧光黄转变为荧光红(365nm紫外光照射下)。在吸收光谱中，加入BChE后探针LAN-bche在575nm处的吸收峰明显增强并伴随着溶液颜色肉眼可见地从淡黄色向红色的转变。以上结果表明探针LAN-bche能够通过荧光法和比色法检测BChE。
本发明所述荧光探针LAN-bche稳定性良好，如良好的光稳定性，25～42℃的温度稳定性、pH稳定性和低毒性能，因此可将其应用于活体检测，如细胞检测和组织检测。
本发明还提供了上述荧光探针LAN-bche的制备方法如图1所示，包括以下步骤：将LAN-OH与环丙甲酰氯混合后进行取代反应，得式I所示的荧光探针LAN-bche。
本发明所述制备方法，优选包括冰浴条件下，将LAN-OH、环丙甲酰氯和绝干二氯甲烷混合，在三乙胺作用下反应，反应产物中包括式I所示的荧光探针LAN-bche。本发明所述LAN-OH优选具有如式II所示的结构；
本发明还提供了上述荧光探针LAN-bche的在制备检测丁酰胆碱酯酶(BChE)的产品中的应用。
本发明所述荧光探针LAN-bche与BChE的响应机理如图2所示，即BChE特异性识别并切断探针LAN-bche的甲酸环丙酯残基释放出荧光团LAN-OH，最终实现对BChE的特异性检测。本发明所述特异性检测优选包括可视化检测，更优选包括通过荧光法和比色法双模式实现对BChE的定量检测，且经实施例验证，所述荧光探针LAN-bche能够在300倍稀释的人血清中精准检测BChE含量。
本发明还提供了上述荧光探针LAN-bche在制备疾病诊断产品中的应用，所述疾病的诊断生物标志物包括丁酰胆碱酯酶。
本发明实施例证实了所述荧光探针LAN-bche即便是在50μM浓度下，对于细胞活性影响不大，且在浓度低于50μM时溶血率约为3.4％，证实所述荧光探针LAN-bche具有极低的生物毒性和较高的生物安全性，可将所述荧光探针LAN-bche应用于细胞成像。本发明还利用所述荧光探针LAN-bche进行细胞成像、组织成像以及内源性BChE检测，证实癌细胞和肝损伤细胞内BChE水平较正常肝细胞下调，推测肝细胞的癌变和损伤导致其合成BChE的能力下降，并能够轻松区分混合细胞中的HepG2肝癌细胞和LO2肝细胞。在活体成像中，探针LAN-bche成功用于活体内BChE的荧光成像，并证实了肝损伤小鼠较正常小鼠体内BChE水平下调，而糖尿病小鼠较正常小鼠上调。
为了进一步说明本发明，下面结合附图和实施例对本发明提供的一种特异性识别丁酰胆碱酯酶的荧光探针LAN-bche及其制备方法和应用进行详细地描述，但不能将它们理解为对本发明保护范围的限定。
本发明实施例中，除非另有说明，否则本工作中使用的所有化学试剂均为购买自药品供应商的分析试剂级，使用时未经进一步提纯。质谱数据通过TSQ QuantumAccessMAX三重四极杆质谱仪(ThermoFisherScientific，美国)获得。环丙甲酰氯、三乙胺、CDCl3、二甲基亚砜、对乙酰氨基酚、Tacrine、RFP、羧甲基纤维素钠盐、链脲佐菌素购买自安耐吉化学有限公司。HeLa细胞、HepG2细胞购买自赛百慷(上海)生物技术股份有限公司。BALB/c-nu小鼠购买自北京华阜康生物科技股份有限公司。血清、全血来自于吉林大学中日联谊医院。所有水溶液均由所有溶液均采用超纯水配制(Milli-Qsystem,Millipore，美国)。
实施例1
根据图1所示流程制备LAN-bche：
(1)化合物1的合成
冰浴条件下，依次向烧杯中加入350mL冰水和6.3mL浓硫酸并充分搅拌，然后将间苯二酚(14.5g，0.13mol)与亚硝酸钠(10.8g，0.15mol)的混合液逐滴加入烧杯，滴加完毕后继续搅拌20min后停止反应，然后将产物进行抽滤、冷冻干燥。最终产物为明黄色固体，不需要进一步提纯即可用于下一步合成(17.93g，96％)。
(2)化合物LAN-OH(式II)的合成
室温下，将化合物1(0.153g，1.1mmol)和1-萘酚(0.144g，1mmol)加入到5mL二氯甲烷中进行充分搅拌。随后将3mL浓盐酸逐滴加入反应瓶，搅拌5h后停止反应。以石油醚和乙酸乙酯(v/v＝5:1)作为流动相，通过硅胶柱层析法进行纯化，化合物LAN-OH为黄褐色固体(130mg，65％)。1HNMR(300MHz,DMSO-d6)δ8.60(d,J＝8.0Hz,1H),8.14(d,J＝7.8Hz,1H),7.85(t,J＝7.5Hz,1H),7.78(d,J＝7.4Hz,1H),7.70(d,J＝8.7Hz,1H),6.85(d,J＝8.8Hz,1H),6.76(s,1H),6.36(s,1H)(图3)。13CNMR(75MHz,DMSO-d6)δ182.42,161.50,151.35,145.32,142.41,132.03,131.33,131.20,131.09,131.01,126.14,125.11,123.82,114.02,105.70,105.64,101.77(图4)。MS(LC-HRMS,m/z)forC16H10NO3 +[M+H]+:calculated,264.0655；found:264.0652(图5)。
(3)化合物LAN-bche(式I)的合成
冰浴条件下，将化合物LAN-OH(0.05mmol，131mg)和环丙甲酰氯(1mmol，80uL)加入10mL绝干二氯甲烷中搅拌10min，随后向其中滴加三乙胺(1mmol，139uL)，滴加完毕后室温搅拌3h。以石油醚和乙酸乙酯的混合物(v/v＝1：1)为流动相，经硅胶柱层析法提纯最终得黄色固体产物(产率83％)。1HNMR(300MHz,CDCl3)δ8.70–8.63(m,1H),8.27(dt,J＝5.6,3.2Hz,1H),7.81–7.71(m,3H),7.14–7.07(m,2H),6.40(d,J＝2.3Hz,1H),1.95–1.79(m,1H),1.26–1.18(m,2H),1.09(dq,J＝4.9,3.4Hz,2H)(图6)。13CNMR(75MHz,cdcl3)δ183.74,172.75,152.56,150.76,146.82,144.30,131.99,131.78,130.35,125.87,124.60,118.82,109.47,107.62,107.58,77.41,76.99,76.56,13.00,9.70(图7)。MS(m/z)for C20H14NO4 +[M+H]+:calculated,332.0917；found:332.0911(图8)。
实施例2
2.1光谱检测过程
将探针LAN-bche溶解于二甲基亚砜(DMSO)制得1mM原液。由10μL探针原液、一定体积浓度的待分析物和一定体积的PBS构成的1mL测试样品溶液分别通过荧光光谱仪和紫外可见分光光度计采集数据。除非特殊说明，否则待测体系在37℃含有1％(v/v)DMSO的PBS缓冲溶液(10mM，pH7.4)中反应70min后用于检测。使用荧光光谱仪测试时，激发光设置为580nm，狭缝为2.5/2.5nm，电压为700V，记录590～750nm范围的发射光谱。使用紫外可见分光光度计测试时，使用1cm石英比色皿记录460～650nm范围的吸收光谱。
在荧光光谱中(图9中A)，在580nm激发光的照射下，加入BChE后探针LAN-bche在642nm处的荧光强度显著增强并伴随着溶液颜色由荧光黄转变为荧光红(365nm紫外光照射下)。在吸收光谱中(图9中B)，加入BChE后探针LAN-bche在575nm处的吸收峰明显增强并伴随着溶液颜色肉眼可见地从淡黄色向红色的转变。表明探针LAN-bche能够通过荧光法和比色法检测BChE。
探究荧光探针LAN-bche以及反应体系的光稳定性。结果如图9中C所示，在580nm的激发光连续80min照射下，探针LAN-bche以及反应体系的荧光强度基本保持不变，这表明探针LAN-bche以及反应体系的光稳定性。随后，探究温度对探针LAN-bche以及反应体系的影响。结果如图9中D所示，在25～42℃温度范围内，探针LAN-bche的荧光强度不随温度的改变而变化，而反应体系的荧光强度随着温度的升高而逐渐增强，证实适当温度范围内温度的升高能加快酶促反应。
探究所述荧光探针LAN-bche与BChE响应随时间的变化规律。结果如图9中E所示，随着时间的进行，探针LAN-bche与不同浓度的BChE反应产生的荧光信号逐渐增强并在70min时达到最大值。最后，探究pH对探针LAN-bche以及反应体系的影响。结果如图9中F所示，在pH5～9范围内，探针LAN-bche的荧光强度基本保持不变，而反应体系的荧光强度随着pH的增大而逐渐增强。综合以上实验结果并考虑到生物体系的环境，接下来的体外实验均在37℃下的PBS(10mM，pH7.4)中反应70min后进行检测。
2.2检出限及荧光量子产率的测定
(1)检出限的测定。通过荧光法和比色法分别采集20个10μM探针LAN-βgal空白样品在642nm处的荧光强度和575nm处的吸光度以计算出标准偏差σ。根据公式DL＝3σ/k计算出探针LAN-bche对BChE的检出限，公式中σ：空白样品的标准偏差；k：标准曲线斜率；DL：检出限。
(2)荧光量子产率的测定。首先制备待测样品和空白样品。10μM探针LAN-bche待测样品：10μL探针原液加入到990μLPBS中(10mM，pH7.4)；10μM荧光团LAN-OH待测样品：10μL荧光团(1mM)加入到990μLPBS中(10mM，pH7.4)；空白样品：1mLPBS(10mM，pH7.4)。待测样品及空白样品的荧光量子产率由稳态瞬态荧光光谱仪(FLS920，EdinburghInstrument)测得(λex＝580nm)。
在最优条件下使用探针LAN-bche对BChE进行定量检测。在荧光法中，在580nm激发光照射下，随着BChE浓度的逐渐增大(0～700U/L)，反应体系在642nm处的发射峰逐渐增强，反应体系荧光强度与BChE浓度(0～200U/L)之间存在良好的线性关系(r＝0.9979)，检出限低至0.056U/L，这表明探针LAN-bche能够通过荧光法定量检测BChE(图10中A和B)。探针LAN-bche对BChE的检测在荧光增强倍数、激发/发射波长及检出限等方面与其他已报道的荧光探针的比较如表1所示，探针LAN-bche表现出明显的优势。在比色法中(图10中C、D)，随着BChE浓度的增大(0～700U/L)，反应体系在575nm处的吸收峰逐渐增强，反应体系的吸光度与BChE浓度(0～200U/L)之间存在良好的线性关系(r＝0.9979)，检出限低至4.92U/L，表明探针LAN-bche能够通过比色法定量检测BChE。
表1探针LAN-bche与其它报道过的荧光探针分析性能的比较
表1中LAN-bche为本发明实施例1中制备得到，其余结构的荧光探针依次来源于：
YOO S,HAN MS.A Fluorescent Probe for Butyrylcholinesterase Activityin Human Serum Based on a Fluorophore with Specific Binding Affinity forHuman Serum Albumin[J].Chem Commun(Camb).2019,55:14574-14577.
CAO T,ZHENG L,ZHANG L,et al.A Highly Butyrylcholinesterase SelectiveRed-Emissive Mitochondria-Targeted Fluorescent Indicator Imaging in LiverTissue of Mice[J].Sensors and Actuators B:Chemical.2021,330
ZHANG W D,ZHANG J M,QIN C Z,et al.A Far-Red/near-InfraredFluorescence Probe with Large Stokes Shift for MonitoringButyrylcholinesterase(Bche)in Living Cells and in Vivo[J].Anal ChimActa.2022,1235:340540.
ZHANG Q,FU C,GUO X,et al.Fluorescent Determination ofButyrylcholinesterase Activity and Its Application in Biological Imaging andPesticide Residue Detection[J].ACS Sens.2021,6:1138-1146.
ZHANG P,FU C,LIU H,et al.And-Logic Strategy for Accurate Analysis ofAlzheimer's Disease Via Fluorescent Probe Lighted up by Two SpecificBiomarkers[J].Anal Chem.2021,93:11337-11345.
2.3酶动力学实验
首先在PBS(10mM，pH7.4)中制备不同浓度的探针LAN-bche(1，2，3，4，5，6，7，8，9，10μM)，然后分别加入200U/L的BChE，立即在10min范围内每1min记录一次荧光强度(λex＝580nm，λemmax＝642nm)，初始反应速率由线性响应范围内的数据确定，根据公式(Michaelis-Menten equation)和(Lineweaver-Burk equation)计算出水解过程动力学参数。
水解过程动力学参数由下式计算:
Michaelis-Menten equation:
Lineweaver-Burk equation:
其中，v：初始反应速率；V：初始反应速率最大值；[S]：底物(探针)浓度；Km：Michaelis常数。
利用Michaelis-Menten方程和Lineweaver-Burk双倒数作图法探究探针LAN-bche与BChE之间的反应动力学。结果如图11所示，在Michaelis-Menten图中(图11中A)，随着探针LAN-bche浓度的逐渐增大，反应初速度呈现出由快速到缓慢的增强效果，根据初速度和探针LAN-bche浓度经换算作出Lineweaver-Burk图(图11中B)，计算出最大速度Vmax＝99.30μM min-1，米氏常数Km＝4.96μM，这表明探针LAN-bche对BChE具有良好的亲和力和高度的敏感性。
为了验证所述荧光探针LAN-bche的特异性，探究包括阳离子、阴离子、氨基酸、小分子、蛋白酶在内的约60种潜在干扰物对探针LAN-bche的影响。结果如图12所示，即使干扰物的浓度已高达1mM，探针LAN-bche对干扰物的响应程度非常低，特别说明乙酰胆碱酯酶(AChE)浓度即使高达1000U/L对BChE几乎无干扰，表明探针LAN-bche能够轻松鉴别AchE与BChE，以上结果证明了探针LAN-bche具有高选择性。
2.4人血清加标回收率实验
首先将人血清样品离心(8000rpm，5min)，然后向稀释300倍的人血清上清液中分别加入25，50，75U/LBChE，最后再加10μM探针LAN-bche，在37℃下孵育70min后用于荧光测试。
结果如表2所示，结果显示在稀释300倍的人血清中加入25，50，75U/LBChE的回收率范围在105.5～108.7％，良好的加标回收率表明探针LAN-bche可以用于人血清中BChE的精准检测。
表33人血清中BChE的加标回收率(n＝3)
2.5分子对接与理论计算
(1)利用Autodock4.0软件对探针LAN-bche与BChE进行分子对接模拟(PDBcode:4fkh)并使用AutodockTools对BChE结构进行处理。在对接探针LAN-bche之前，分配了AD4原子类型并计算了Gasteiger电荷。所有可旋转键均设置为主动扭转。对接周期、探针LAN-bche和BChE参数均按缺省值配置。通过PyMOL2.3.3和DiscoveryStudio2020软件对对接模型进行可视化。
(2)使用Gaussian16软件在B3LYP(GD3BJ)/def2-SVP水平上，利用密度泛函理论(DFT)和时变DFT(TDDFT)对探针LAN-βgal的最佳几何结构和电子结构进行了计算并利用Multiwfn3.7软件对所得数据进行进一步分析。
利用质谱检测、HPLC分析以及抑制剂实验探究探针LAN-bche与BChE的传感机理。在质谱测检测中，探针LAN-bche的质谱峰m/z为331.9298(图8)，荧光团LAN-OH的质谱峰m/z为264.0646(图13)，而反应体系的质谱峰m/z为264.0649(图14)，这与荧光团的质谱峰数据相吻合。
HPLC分析结果如图15所示，探针LAN-bche的出峰时间为1.0min，荧光团LAN-OH的出峰时间为2.7min，而反应体系在1.0min和2.7min均有峰出现，其中出峰时间2.7min与荧光团的出峰时间相吻合。
在抑制剂实验中，抑制剂Tacrine的加入对探针LAN-bche和荧光团LAN-OH的荧光强度均无影响，而明显抑制了反应体系的荧光强度(图16)。综合以上结果，探针LAN-bche与BChE的响应机理如图2所示，即BChE特异性识别并切断探针LAN-bche的甲酸环丙酯残基释放出荧光团LAN-OH，最终实现对BChE的特异性检测。
探针LAN-bche与BChE的分子对接方式如图17所示，探针LAN-bche与BChE中的2个氨基酸(His438，Ser198)形成2个氢键，结合能为-9.7kcal/mol，表明探针LAN-bche与BChE具有较强的结合亲和力。对探针LAN-bche和荧光团LAN-OH的分子轨道进行理论计算。探针LAN-bche的HOMO和LUMO都主要位于整个荧光团中，虽然在荧光团LAN-OH的HOMO和LUMO上没有电子密度的再分布，但对应典型ICT的S1到S0的跃迁(625.7nm,f＝1.1193)导致探针LAN-bche与BChE响应后产生强烈的荧光。荧光团LAN-OH(2.58eV)的能隙低于探针LAN-bche(3.16eV)，可以合理地证明BChE的加入使探针LAN-bche的吸收波长的红移。以上分子对接模拟和理论计算的结果支持了图2中所提出的探针LAN-bche对BChE的传感机理。
实施例3
3.1细胞实验
3.1.1细胞培养
所有细胞均在含有10％胎牛血清(FBS)、100U/mL青霉素和100μg/mL链霉素的RPMI1640中培养并保存在37℃的含5％二氧化碳/95％空气的培养箱中。
3.1.2细胞毒性分析
采用CCK-8法测试探针LAN-bche对HepG2细胞的毒性。首先向96孔板每孔中加入100μLHepG2细胞悬液(105个细胞/mL)并保存在37℃含5％二氧化碳/95％空气的培养箱中24h，然后在每孔中加入10μL不同浓度(5，10，20，30，50μM)的探针LAN-bche并孵育24h。接下来在每孔中加入10uLCCK-8溶液并孵育1h，最后使用酶标仪在450nm波长处测定每孔的吸光度。
结果如图18所示，即使探针LAN-bche浓度高达50uM细胞的存活率仍然很高，这证明了探针LAN-bche的低细胞毒性。
3.1.3溶血分析
将1mL健康人外周血全血离心(3000rpm，5min)去除血浆，然后在相同条件下用5mL生理盐水洗涤5遍，5mL生理盐水重悬后冰上待用。将不同浓度(5、10、20、50、100、200μM)的探针LAN-bche溶液、红细胞悬液和生理盐水加入离心管后轻轻吹打混匀并置于37℃水浴中避光3h。取出离心管离心(3000rpm，5min)后进行拍照记录。然后从每个试管中取出300uL(100uL/孔，3孔)，在540nm处检测吸光度。各组溶血率按以下公式计算：
溶血率(％)＝(样品-阴性)/(阳性-阴性)
注释：PBS为阴性对照，H2O为阳性对照
不同(5，10，20，50，100，200μM)的探针LAN-bche对红细胞的溶血率，结果如图19所示，探针LAN-bche浓度低于50μM时溶血率约为3.4％，这说明探针LAN-bche具有较高的生物安全性。
3.1.4细胞成像
(1)时间成像实验。准备9组LO2细胞并分别用10μM探针LAN-bche孵育，并记录不同时间时刻细胞的成像情况。
结果如图20所示，细胞内荧光信号随时间逐渐增强并在1h左右达到最大值，因此在后续细胞成像实验中将探针LAN-bche孵育细胞的时间设定为1h。
(2)细胞内源性BChE成像。第一组HepG2细胞和LO2细胞作为对照组。第二组HepG2细胞和LO2细胞用10μM探针LAN-bche孵育1h后成像。第三组HepG2细胞和LO2细胞先用100uM抑制剂Tacrine孵育1h，再用10μM探针LAN-bche孵育1h后成像。
结果如图21所示，LO2细胞和HepG2细胞均产生荧光信号，但LO2细胞内的荧光信号明显强于HepG2细胞，这可能是因为肝细胞的癌变导致其合成BChE的能力下降。而抑制剂Tacrine的加入明显抑制了LO2细胞和HepG2细胞内的荧光信号强度。以上结果表明探针LAN-bche能够成功用于细胞内BChE的成像。
(3)肝损伤细胞内源BChE成像实验。第一组HepG2细胞和LO2细胞作为对照组。第二组HepG2细胞和LO2细胞用10μM探针LAN-bche孵育1h后成像。第三组HepG2细胞和LO2细胞预先用1mM对乙酰氨基酚(APAP)孵育12h，再用10μM探针LAN-bche孵育1h后成像。
利用探针LAN-bche监测APAP诱导的肝损伤细胞内BChE水平的能力，结果如图22所示，与LO2细胞和HepG2细胞相比(b和e)，经APAP预处理的肝损伤组细胞(c和f)显示出更弱的荧光信号，这可能是因为细胞受损导致其合成BChE的能力下降。以上结果证明了探针LAN-bche能够用于监测肝损伤细胞内BChE水平的波动。
(4)混合细胞成像实验。首先用5μMHoechst33342核酸染料孵育LO2细胞5min，然后用1mLPBS(10mM，pH7.4)洗涤3次，最后加入HepG2细胞共培养。待混合细胞培养好后，加入10μM探针LAN-bche孵育1h后成像。
结果如图23所示，经核酸染料Hoechst33342染核的LO2细胞在蓝色通道有明显荧光信号而HepG2细胞无荧光信号(图23中B)，在经探针LAN-bche处理后，LO2细胞在红色通道有明显的荧光信号而HepG2细胞显示出极其微弱的荧光信号(图23中C)，在明场、蓝色通道和红色通道的复合通道(图23中D)中可以更清楚地观察到LO2细胞荧光信号明显强于HepG2细胞。以上结果表明探针LAN-bche能够轻松区分混合细胞中的LO2细胞和HepG2细胞。
3.2活体实验
3.2.1小鼠模型的建立
动物实验程序由吉林大学动物实验伦理委员会批准。
(1)荷瘤小鼠模型(HepG2肿瘤)：首先将1×107HepG2细胞悬浮于300μLPBS(10mM，pH7.4)中，然后将其接种于裸鼠上肢腋下，15天后获得肿瘤模型。
(2)APAP诱导的肝损伤小鼠模型：首先将APAP溶解于温生理盐水中配制15mg/mL的APAP溶液，然后将禁食过夜的裸鼠腹腔注射300mg/kgAPAP溶液，10h后模型构建完成。
(3)糖尿病小鼠模型：首先新鲜配制pH4.5的50mg/mL柠檬酸链脲佐菌素缓冲液(STZ的柠檬酸缓冲液)。裸鼠禁食一夜后腹腔注射/尾静脉注射150mg/kg的STZ的柠檬酸缓冲液，3天后成功诱导糖尿病小鼠，用血糖仪测量血糖>13mM诊断为糖尿病。
(4)糖尿病治疗小鼠模型：首先按照(3)的步骤构建糖尿病裸鼠。接下来给糖尿病小鼠连续7天灌胃二甲双胍溶液(100μL，200mg/kg)。
所有活体肿瘤成像实验均在IVISLuminaLTSeriesIII小动物成像系统上进行，激发波长为560nm，滤光片为650nm。
3.2.2活体成像
将小鼠分为对照组(正常小鼠)、肝损伤组(APAP诱导的肝损伤)、糖尿病组以及治疗组(糖尿病治愈)等四组，分别对四组小鼠尾静脉注射探针LAN-bche(200μM，100μL)，记录不同时刻小鼠成像情况。
结果如图24所示，随着时间的进行(0-180min)，对照组(图24中A)小鼠在肝部、腹部显示出明显的强荧光信号，而APAP诱导的肝损伤组(图24中B)小鼠相对于对照组荧光信号明显减弱，这表明肝损伤小鼠体内BChE水平较正常小鼠明显下调，推测是因为肝损伤后肝脏合成BChE的能力遭到破坏。糖尿病组(图24中C)小鼠相对于对照组显示出更强的荧光信号，而治疗组(图24中D)小鼠相对于糖尿病组显示出较弱的荧光信号，这表明糖尿病小鼠体内BChE水平较正常小鼠明显上调。以上结果表明：探针LAN-bche可用于活体内BChE水平的可视化，辅助肝损伤以及糖尿病的诊断。
尽管上述实施例对本发明做出了详尽的描述，但它仅仅是本发明一部分实施例，而不是全部实施例，人们还可以根据本实施例在不经创造性前提下获得其他实施例，这些实施例都属于本发明保护范围。
Claims (2)
1.一种特异性识别丁酰胆碱酯酶的荧光探针LAN-bche在制备疾病诊断产品中的应用，所述疾病的诊断生物标志物为丁酰胆碱酯酶，所述疾病诊断产品为可视化诊断产品，所述疾病诊断产品基于荧光法或比色法完成可视化诊断；
所述疾病为肝脏疾病和/或糖尿病；
所述荧光探针LAN-bche的分子结构如式I所示，
2.根据权利要求1所述应用，其特征在于，所述疾病诊断产品的诊断样本为血清。
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