真菌毒素是真菌产生的一类具有高毒性的次级代谢产物,广泛分布在农作物、食品和饲料中,不仅会导致食品霉败变质、营养物质损失、降低品质,还会通过对人和动物机体内DNA、RNA、蛋白质和各种酶类的合成抑制以及对细胞结构的破坏而引起人类和动物的急性或慢性中毒[1-2]。据联合国粮农组织估算,全球每年约有25%的农产品受到真菌毒素的污染,所造成的经济损失高达数千亿美元。因此,建立高效快速的真菌毒素检测方法受到越来越多的关注。目前针对真菌毒素常用的检测方法有液相色谱-串联质谱(liquid chromatography-mass spectrometry,LC-MS)[3]、薄层色谱(thin layer chromatography,TLC)[4]和高效液相色谱(high performance liquid chromatography,HPLC)[5]等。虽然这些方法具有灵敏度高、分离效能高等优点,但由于需要繁琐的前处理步骤、昂贵的仪器和专业的操作人员,因而并没有广泛应用。免疫分析技术具有检测速度快、特异性好等优点,可以实现大批量样品的快速检测,但价格昂贵、试验结果易出现假阳性,而且真菌毒素大部分属于半抗原,因此其作为免疫原获得抗体比较困难[6]。
分子印迹技术(molecular imprinting technology,MIT)是基于费希尔模拟的“分子钥匙和锁”原理设计的一种人工合成的分子识别技术[7]。MIT利用模板分子(待测物)选择合适的功能单体使之形成可逆的复合物,加入交联剂和引发剂后在特定的条件下反应形成分子印迹聚合物(molecularly imprinted polymers,MIPs),后通过物理或化学方式将模板分子洗脱下来,即可得到具有选择性好、热稳定性高、制备简单和成本低廉等优良性能的仿生抗体。得到的识别位点能够特异性结合该模板分子,进而可以实现对后续的待测物进行高灵敏特异性检测[8-9]。分子印迹技术所制备的分子印迹聚合物具有特异识别性、实用性、鲁棒性、成本低、灵敏度高、可设计性强和稳定性高等特点,广泛应用于食品安全检测领域[10-11]。分子印迹技术的基本原理如图 1 所示[8]。
图1 分子印迹技术的基本原理
Fig.1 Basic principle of molecular imprinting technology
近年来,出现了一些新型的基于功能性纳米材料标记的分子印迹技术,突破了传统分子印迹技术在灵敏度和稳定性上的不足,为真菌毒素高效快速的分析检测以及样品前处理提供了新方法,也为食品危害因子的有效控制提供了新思路。本文对基于功能性纳米材料的分子印迹技术在真菌毒素检测中的应用进行综述,包括金纳米粒子、碳纳米管、金属-有机骨架、荧光材料(如量子点、碳量子点和上转换纳米粒子)和磁性纳米材料等,以期为新型功能性纳米材料的分子印迹技术在真菌毒素分析和检测领域的应用提供参考。
分子印迹聚合物(MIPs)的制备具有操作简单、直接等特点。目前,MIPs主要有以下几种制备方法:本体聚合、乳液聚合、悬浮聚合、溶胶-凝胶聚合、表面分子印迹、沉淀聚合等。不同制备方法的优缺点对比见表1。
表1 MIPs不同制备方法对比
Table 1 Comparisions of different preparation imprinting methods of MIPs
制备方法 优点 缺点 参考文献本体聚合 操作简单 前处理费时又费力且聚合物收率低[12]乳液聚合 反应体系与产品粒径均一且传热均匀体系组成复杂、合成条件苛刻且产品产率低下[13-14]悬浮聚合 聚合物形状规则且结合位点不易被破环,粒径易于调整单分散性很低,受分散剂影响较大[15-17]溶胶-凝胶聚合有效识别能力强,再生性能稳定且产物均匀、刚性好聚合时间较长,酸碱产物不同[18-20]表面分子印迹吸附能力强且平衡时间短操作过程较复杂 [21-22]沉淀聚合 操作简单、所得粒子表面整洁需大量模板分子且仅限于高丰度模板[23-24]
金、银等贵金属纳米粒子结合了纳米材料和金属的特性,具有优异的电学、光学性质以及高度的生物相容性[25]。例如金纳米粒子(gold nanoparticles,AuNPs)具有生物相容性良好、比表面积大且化学物理性质稳定的优点,具有出介导信号放大的作用,将其与MIPs技术相结合用于真菌毒素的检测具有很好的研究意义[26]。
Zhang 等[27]利用 3(2,2′-联吡啶)-钌(II)[tris(2,2′-bipyridine)-ruthenium(II),Ru(bpy)32+]掺加二氧化硅纳米粒子(Ru@SiO2NPs)构建了一种新型分子印迹电化学发光传感器,并用于牛奶和玉米样品中伏马毒素B1(fumonisin B1,FB1)的高灵敏检测。该研究基于AuNPs优异的局域表面等离子体共振效应和电化学效应,实现提高分析灵敏度的目的。此传感器对FB1的检出限能够达到0.35pg/mL,线性范围为0.001ng/mL~100ng/mL。AKGÖNÜLLÜ等[28]以黄曲霉毒素B1(aflatoxin B1,AFB1)和N-甲基丙烯酰苯丙氨酸为模板分子和功能单体进行预复合,然后在表面等离子共振(surface plasmon resonance,SPR)金芯片表面涂覆了含AuNPs的分子印迹聚合物,制备了一种新型的纳米印迹传感器,用以检测玉米样品中的AFB1。AFB1印迹纳米传感器显示出很宽的线性范围,介于0.000 1 ng/mL和10.0 ng/mL之间,检测限为1.04 pg/mL。实现了对多种食品样品中AFB1高灵敏度和高选择性的检测。Gu等[29]开发了一种AuNPs掺杂分子印迹层和共价有机骨架复合物(COFs-AuNPs)的石英晶体微天平(quartz crystal microbalance,QCM)传感器,用于花生、开心果、水稻、小麦样品中AFB1的检测。COFs-AuNPs复合物提供了较大的比表面积,具有灵敏度高和选择性强的特性。该传感器线性范围为0.05 ng/mL~75 ng/mL,检测限为2.8 pg/mL,实际样品回收率介于87.0%~101.7%之间。Fang等[30]将邻氨基噻吩电聚合在金纳米颗粒复合介孔碳(AuNPs@CMK-3)修饰的金电极表面,构建了一种新型三维分子印迹石英晶体微天平(quartz crystal microba-lance,QCM)传感器用于谷物样品痕量桔霉素(citrinin,CIT)检测。由于AuNPs@CMK-3功能复合物的3D结构和大的比表面积有利于增加有效印迹位点的数量并随后提高传感器的灵敏度。因此,AuNPs@CMK-3功能复合物可充当信号放大器。该方法检测限为1.8×10-9mol/L,表现出良好的选择性识别能力、抗干扰能力、重现性好和稳定性高等特点。Jiang等[31]以氨基噻吩功能化的AuNPs为模板分子,电聚合制备了一种用于检测杏仁、巴西坚果、榛子、开心果、无花果干中AFB1的电化学分子印迹传感器,示意图如图2所示[31]。该分子印迹传感器的线性范围很宽,在3.2×10-15mol/L~3.2×10-6mol/L 之间,定量限为 3×10-15mol/L,表现出灵敏度高重复性好的特点,有望成为食品中AFB1选择性电化学检测的有效方法。
图2 基于AuNPs的黄曲霉毒素B1分子印迹传感器示意图
Fig.2 Schematic diagram of aflatoxin B1 molecularly imprinted sensor based on AuNPs
碳纳米管(carbon nanotubes,CNTs)是一种重要的功能性纳米材料,具有轻质、刚性、比表面积大、高拉伸机械强度、导热性良好、抗机械损伤和化学性质独特等优点,广泛应用于医疗、生物工程、电子和环境保护等领域[32-33]。CNTs分为单壁碳纳米管和多壁碳纳米管(multi walled carbon nanotubes,MWCNT),前者仅仅由一层圆柱型石墨烯片构成,后者则含有一层以上石墨烯片层。其中多壁碳纳米管具有独特的管状结构和极高的表面积,并且具有能够储存各种元素和化学物质的多孔结构,使其对真菌毒素的吸附具有选择性,从而在真菌毒素的检测中具有广泛的应用[34]。Pacheco等[35]通过用MWCNT和MIPs修饰玻碳电极(glassy carbon electrode,GCE),制备了一种用于检测赭曲霉毒素A(ochratoxin A,OTA)的新型电化学传感器,示意图如图3所示[34]。
图3 MWCNT-MIPs制备示意图
Fig.3 Preparation of MWCNT-MIPS
MWCNT的应用增加了传感器的电导率和表面积,大大提高了传感器的灵敏度。MIP/MWCNT/GCE传感器使用差分脉冲伏安法时,检测限和定量限分别为1.7 μg/L 和 5.7 μg/L。由于 MIPs/MWCNT/GCE 传感器易于制造且易于使用,已成功用于加标啤酒和葡萄酒样品中OTA的测定,回收率在84%~104%之间,且无需样品预处理步骤。
金属-有机骨架(metal-organic frameworks,MOFs)是一类由金属离子或金属离子簇与有机配体连接而成的相对较新的多孔晶体杂化有机-无机材料[36]。MOFs通过多齿有机配体和金属离子的自组装,合成具有类沸石网络结构的金属有机骨架[37]。在MOFs中,有机配体和金属离子或团簇的排列具有明显的方向性,可以形成不同的框架孔隙结构,从而表现出不同的吸附性能、光学性质、电磁学性质等。虽然MOFs材料在潮湿环境中的稳定性及吸附性能均会明显降低,但由于MOFs具有超高孔隙率、稳定骨架、无毒性、大的内表面积和化学功能高等优点,在现代材料学方面呈现出巨大的发展潜力和发展前景。
BAGHERI等[38]将MIPs的选择性识别特性与新型银纳米粒子/片状锌基 MOFs纳米复合材料(Ag-NPs@ZnMOFs)突出的过氧化物酶活性以及灵敏的荧光检测系统相结合,设计了一种可靠的展青霉素(patulin,PAT)探针,用于复杂水生环境或苹果汁中PAT的检测。该复合材料具有良好的稳定性、较大的比表面积和较高的选择性吸附亲和力。在PAT存在的情况下,荧光强度在 0.1 μmol/L~10 μmol/L的范围内与其浓度成比例地降低,检测限为0.06 μmol/L。该方法能够在复杂介质中选择性地检测PAT,而没有来自模拟化合物的显著干扰作用。Liang等[39]将MIPs与氨基功能化的锆基金属-有机骨架相结合(UiO-66-NH2@MIPs),制备了UiO-66-NH2@MIPs型表面印迹聚合物。利用MOFs的特殊识别特性、多孔性和化学修饰性,制备了新型吸附剂,并成功地用于粮食中黄曲霉毒素(aflatoxin,AFs)的定量分析。HPLC测定结果表明经过该吸附剂处理的样品具有良好的线性范围(0.20 μg/kg~45 μg/kg)、低检测限(90 μg/kg~130 μg/kg)和较好的回收率(74.3%~98.6%),证明该新型吸附剂具有良好的选择性和可重用性。Du等[40]通过利用发光金属-有机骨架建立了一种基于分子印迹聚合物的荧光传感器,可高灵敏度检测小麦中玉米赤霉烯酮(zearalenone,ZEN)。结果表明,该方法的检出限为0.018 mg/L,且具有良好的回收率(95.53%~98.01%)。该传感器表现出稳定性高,选择性好的优点。
MOFs材料受热易分解,因此可将其高温煅烧碳化制备稳定的纳米多孔碳(nanoporous carbon,MNPC)材料,具有重要的研究意义[41]。Wu等[42]采用原子转移自由基沉淀聚合法,在MNPC核表面包覆MIPs,其以3-香豆素羧酸乙酯为假模板,得到对AFs具有特异识别能力的MIPs。MNPC具有大孔体积,可以方便、有效地覆盖聚合物层。利用MNPC@MIPs提取AFs,再用高效液相色谱法测定AFs的含量。在最佳条件下,加标玉米样品中AFs的检测限通常为0.05ng/mL~0.07ng/mL,回收率为 75.1%~99.4%,相对标准偏差为 1.7~5.1(n=6)。
荧光纳米粒子分子印迹聚合物是指在聚合物中导入具有特异性识别能力的荧光信号,使待测物能够被特定的荧光检测机制所识别,具有灵敏度高,反应迅速等优点。目前常见的具有可操作性的荧光纳米粒子主要有量子点、碳量子点、上转换纳米材料等。
2.4.1 量子点分子印迹聚合物在真菌毒素检测中的应用
量子点(quantum dot,QDs)作为一种越来越受关注的荧光纳米粒子,是一种纳米级别的半导体。因其具有独特的光学和电子特性,如具有宽激发光的清晰发射带和对光致漂白分子印迹聚合物的强耐受性,从而体现出对称发射分布、高量子产率和高灵敏度等突出优点,被广泛应用于各种检测中[43-44]。但传统量子点多含有重金属元素,如汞、镉等,操作不当容易对人类和环境造成一定的危害,因此在食品检测中应合理利用QDs[45]。
MADURANGIKA等[46]用MIPs纳米传感器包覆Mn掺杂ZnS量子点制备了一种简单的室温磷光纳米传感器,并应用于鱼饲料中AFs的测定,样品检出限为3.56 μg/kg。该方法具有吸附能力强、成本低、灵敏度高、选择性好等特点。Zhang等[47]采用表面分子印迹溶胶-凝胶法合成了Mn掺杂ZnS量子点纳米传感器,用于苹果汁中PAT的选择性测定。MIPs-QDs比非印迹聚合物具有更高的选择性、吸附容量和传质速率,在竞争性真菌毒素及其类似物中,对PAT有特异性识别能力。MIPs-QDs对PAT的识别线性范围为0.43 μmol/L~6.50 μmol/L,方法检测限为 0.32 μmol/L,相关系数(R2)为0.994 5。Guo等[48]以3-氨丙基三乙氧基硅烷为功能单体,正硅酸乙酯为交联剂,并将分子印迹技术与量子点技术相结合,合成了一种新型、灵敏的荧光传感器,用以检测食品和中药样品中的AFB1。该试验在最佳条件下,MIPs@CdTe QDs基于荧光共振能量转移机制,其检测样品的相对荧光强度与AFB1浓度在80ng/g~400 ng/g范围内呈良好的线性关系,检测限为4 ng/g。Chmangui等[49]将合成的MIPs材料锚定在聚乙二醇-锰 (polyethylene glycol manganese,PEG-Mn) 掺杂的ZnS量子点(QDs)表面,建立了识别测定非乳饮料中AFs的荧光探针。制备的MIPs-QDs复合物对AFs具有很高的亲和力和选择性,该方法检测限和定量限分别为0.016 mg/L和0.053 mg/L。
石墨烯量子点(graphene quantum dots,GQDs)是石墨烯片的小型化衍生物,其可以分离界面产生的电荷,并向电极提供电荷传输路径[50]。GQDs是一类新型的碳纳米材料,与传统的量子点相比,具有毒性低、生物相容性好等优点[51]。目前,制备石墨烯量子点的方法大致可以分为两种:尺寸调控和表面化学反应,前者是将石墨烯、碳纤维、碳纳米管等含大量石墨烯结构的碳物质通过物理方法、化学方法或电化学方法进行分割得到石墨烯量子点;后者是在石墨烯量子点表面修饰羟基、羰基和羧基等基团或掺杂氮、硫、硼等杂原子,以改变石墨烯量子点的性质[52-53]。Akyıldırım 等[54]以石墨烯量子点(GQDs)修饰钯纳米粒子(palladium nanoparticles,PdNPs)的GCE为基础,制备了用于桔霉素(citrinin,CIT)分析的功能性纳米粒子MIPs。纳米传感器的线性范围为 1.0×10-9mol/L~5.0×10-9mol/L,检出限为2.0×10-10mol/L,其能够快速灵敏的检测鸡蛋样品中的CIT,且材料消耗少。
2.4.2 碳量子点分子印迹聚合物在真菌毒素检测中的应用
碳量子点(carbon quantum dots,CQDs)是一种新兴量子尺寸(10 nm以下)的碳纳米材料,根据前驱体和制备方法的不同,CQDs的制备方法可分为自上向下法和自下而上法,前者主要是将碳源中的大分子分离并石墨化,使大分子变成小的有机物,然后碳化形成CQDs;后者则是小分子聚合形成大分子,然后碳化形成CQDs[55]。CQDs由于具有绿色合成、导电性显著、易于功能化、低毒、生物相容性好和成本低等优点,已被应用于分子印迹领域[56]。
Shao等[57]合成了高发光的CQDs,然后通过非水解溶胶-凝胶法将其封装在硅基基体中,制备了分子印迹荧光猝灭粒子(molecularly imprinted fluorescence quenching particles,MIFQP)。所制备的印迹粒子对谷物样品中的ZEN不仅具有优异的分子识别能力,而且具有良好的光稳定性和明显的模板结合诱导荧光猝灭作用。在优化条件下,MIFQP的荧光强度与ZEN的浓度成反比,MIFQP传感器对玉米样品中的ZEN检测范围在0.02 mg/L~1.0 mg/L之间,检测限为0.02 mg/L,回收率为78%~105%,相对标准偏差小于20%,具有实际应用的潜力。Liang等[58]采用碳量子点修饰的模拟分子印迹聚合物(carbon quantum dots-coated dummy molecularly imprinted polymer,CDs-DMIP) 整体柱预处理,结合高效液相色谱-荧光检测(high performance liquid chromatography fluorescence detection,HPLC-FLD),建立了测定花生样品中AFB1的新方法。该试验对花生样品的检测限和定量限分别为0.118ng/mL和0.393ng/mL。在优化条件下,富集倍数可达71倍以上,CDs-DMIPHPLC-FLD法可灵敏地测定花生样品及其它样品中的AFB1。
2.4.3 上转换纳米材料分子印迹聚合物在真菌毒素检测中的应用
上转换发射荧光是指两个或多个光子的顺序吸收导致光在较短波长(通常在可见范围内)发射的非线性光学过程,而不是激发波长(红外或近红外),这意味着上转换纳米粒子(upconversion nanoparticles,UCNPs)遵循反斯托克斯定律,可以吸收两个或多个低能光子发射高能光子[59]。与传统的荧光材料如荧光染料和半导体量子点相比,上转换纳米粒子具有发射光谱清晰、寿命长、斯托克斯位移大、光稳定性强、自发荧光背景低和毒性低等突出特性,因此越来越受到人们的关注[60-61]。
Liu等[62]基于MIPs的高选择性和UCNPs的荧光特性,制备了具有特异性和荧光信号的MIPs,以识别真菌产生的次生代谢产物杂色曲霉素(aspergillus variegatus,ST),在 0.05 mg/L~1.0 mg/L 范围内,荧光印迹聚合物的荧光强度与浓度呈良好的线性关系,检出限为0.013 mg/L。实际样品大米、玉米和大豆的回收率分别为83.8%~88.8%、82.1%~87.5%和 80.6%~89.2%,表明了该方法的可行性。Yan等[63]合成了一种基于UCNPs的新型MIPs,最终的复合物结合了MIPs的高选择性和UCNPs的高荧光强度的优点,对OTA具有选择性和敏感性。结果表明在最优条件下,当OTA浓度为0.05 mg/L~1.0 mg/L时,荧光印迹聚合物的荧光猝灭程度与OTA的浓度呈现良好的线性关系。OTA的荧光传感检测方法的最低检出限为0.031 mg/L。玉米、大米和饲料中OTA的回收率范围分别为88.0%~91.6%、80.2%~91.6%和89.2%~90.4%。
Fe3O4具有无毒、制备简单和吸附动力学快等特点,近年来,以Fe3O4微球为磁性成分的磁性分子印迹聚合物在真菌毒素检测领域引起了广泛关注。通过在磁性纳米粒子表面上合成MIPs壳,制备了磁性分子印迹聚合物(magnetic molecularly imprinted polymers,MMIPs),其与MIPs相比,MMIPs具有较高的吸附容量和优异的磁性,在目标组分的分离或检测中具有广泛的应用前景[64-65]。
Hu等[66]在磁纳米粒子表面沉积了一种聚多巴胺的分子印迹聚合物(Fe3O4@PDAMIPs),其是一种高效特异的吸附剂,可用于各种赭曲霉毒素的提取。在最佳条件下,赭曲霉毒素A、赭曲霉毒素B和赭曲霉毒素C的校正曲线分别在0.01ng/mL~1.0ng/mL、0.02ng/mL~2.0 ng/mL和0.002 ng/mL~0.2 ng/mL范围内呈线性关系。大米和葡萄酒样品的检出限在1.8 pg/mL~18 pg/mL之间,加标回收率为71.0%~88.5%,相对标准偏差为2.3%和3.8%,此外,MIPs可多次重复使用,节约成本。Huang等[67]以Fe3O4为磁芯,改性埃洛石纳米管为载体,选择性印迹聚合物为壳,制备了MMIPs,并实现了对谷物样品中的ZEN的提取和富集。方法的线性范围为10 ng/mL~200 ng/mL,检测限和定量限分别为2.5 ng/mL和8 ng/mL。玉米样品的ZEN加标回收率在74.95%~88.41%之间。Turan等[68]在磁性纳米粒子表面合成了高选择性的MIPs,实现了对葡萄汁样品中OTA的快速检测。结果表明,MMIPs对OTA具有吸附速度快、吸附容量大、选择性高等特点。加标葡萄汁样品中OTA的回收率为97.1%~97.4%。MMIPs对OTA靶点具有很高的亲和力,在食品样品的纯化方面有很大的应用潜力。Fu等[69]采用表面印迹技术制备MMIPs,将其与高效液相色谱-荧光检测器(HPLC-FLD)结合,快速测定谷物中ZEN。在实际样品检测中,检出限和定量限分别为0.4 ng/kg和0.9 ng/kg。样品回收率为90.56%~99.96%。结果表明,MMIPs对ZEN有很好的选择性,适合于谷物中ZEN的测定。Díaz-Bao等[70]将分子印迹技术与磁性粒子相结合,开发了一种快速简便的制备磁性分子印迹搅拌棒(magnetic molecularly imprinted stirbars,MMIP-SB)的方法。该试验以MMIP-SB为常规搅拌棒,结合高效液相色谱和质谱法测定奶粉(婴儿配方食品)中的黄曲霉毒素M1和谷类婴儿食品中的黄曲霉毒素B1、黄曲霉毒素B2、黄曲霉毒素G1和黄曲霉毒素G2。结果表明,黄曲霉毒素M1、黄曲霉毒素B1、黄曲霉毒素B2、黄曲霉毒素G1和黄曲霉毒素G2的平均回收率分别为60%、43%、40%、44%和39%,相对标准偏差小于10%。Fu等[71]以Fe3O4纳米粒子为载体,采用表面印迹法制备MMIPs,将其与HPLC相结合,快速测定果汁中的PAT。在实际样品应用中,检出限和定量限分别为3μg/kg和10μg/kg,样品回收率为86.44%~95.50%。结果表明,MMIPs对PAT具有良好的分离性能,适合于实际样品中PAT的测定。Cavaliere等[72]以槲皮素为假模板合成MMIPs,从谷物粉中选择性提取ZEN。采用高效液相色谱-串联质谱联用法测定分析物,ZEN回收率大于95%,定量限为0.14 ng/g,比欧洲法律规定的大多数谷物的最大限值低约500倍。
近年来,真菌毒素残留问题受到人们的高度关注,基于新型纳米粒子标记的分子印迹技术将具有一定特性的功能性纳米粒子与分子印迹技术相结合,极大的提高了MIPs识别待测物的灵敏度和吸附性,在真菌毒素检测方面得到广泛应用。新型纳米粒子标记分子印迹技术是将具有一定特性的功能性纳米粒子与分子印迹技术相结合,极大的提高了MIPs识别待测物的灵敏度和吸附性。为了让功能性纳米粒子标记的MIT更好地应用于食品与饲料中真菌毒素的检测,未来的研究方向可能会集中在以下领域:1)真菌毒素本身具有剧毒和价格昂贵等特性,只能通过其结构类似物作为替代模板进行分子印迹聚合物的制备,需解决聚合物特异识别能力较低的问题;2)开发能够同时检测多种待测物的功能性纳米粒子分子印迹传感器,以求实现对大量样品进行快速现场筛查;3)开发新型功能单体、交联剂、引发剂,使得模板分子更易洗脱;4)将功能型纳米粒子与MIPs相结合,进一步实现真菌毒素的快速、高灵敏检测,如:氧化石墨烯与金属氧化物合成用作吸附剂的纳米复合材料,以此来解决氧化石墨烯可靠性和可重复性以及非特异性差的问题,解决MOFs材料在潮湿环境中性能差的问题,解决上转换给体的寿命易缩短和上转换发光易猝灭等问题。
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