CN111867603A - 检测癌细胞的装置与方法 - Google Patents
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Abstract
本发明是关于一种用以检测癌细胞的微流体芯片，尤其是一生物样本中的胆管癌细胞。本发明也涵盖一种使用本发明的微流体芯片来检测一生物样本中的胆管癌细胞的方法。
Description
发明人或共同发明人依37CFR 1.77(B)(6)出具的先前公开事实声明
本申请部分内容已被发明人为蔡玮纯(Tsai,Wei-Chun)及李国宾(Lee,Gwo-Bin)公开于“以八糖磺化乙酰肝素来检测胆管癌细胞的微流***(A Microfluidic System forDetection of Cholangiocarnoma Cells by Using Heparan SulfateOctassaccharide)”文章中。此篇文章是发表于2017年4月9日至4月12日于美国洛杉矶市举办的第12届IEEE世界年会中，其内容是由本发明申请中2位成员所公开，该公开内容全文在此并入本文作为参考。
相关申请
本申请主张依据美国第35号法典第119(e)条于2018年3月1日提出申请的临时申请第62/636,910号的申请日为其优先权日，该临时申请的全部内容并入本文作为参考。
发明背景
1.技术领域
本发明整体来说是关于一种用以检测生物样本中的癌细胞的装置与方法。
2.背景技术
胆管癌(cholangiocarcinoma，CCA)为一种发生于胆管的原发性恶性肝脏肿瘤，其预后相对恶劣，确诊后的五年存活率低于10％。现今的影像学技术：包括超声波检查、核磁共振与计算机断层检查，与利用活体组织切片或细胞学检查等病理诊断法为胆管癌的主要诊断工具。但这些方法皆因肿瘤大小、位置、与病灶的限制，而难以在胆管癌早期发现。最常用于诊断的胆管癌的肿瘤标记为糖抗原19-9(carbohydrate antigen 19-9，CA19-9)与癌胚抗原(carcinoembryonic antigen，CEA)。然而，CA19-9和CEA在临床应用中的敏感性和特异性仍然不令人满意。
因此，相关领域亟需一种可用以检测早期胆管癌的新颖生物标记，以利早期诊断与治疗胆管癌。
发明内容
本发明的发明人意外性地发现一些新颖的磺化八糖可作为可结合到胆管癌细胞的表面的生物标记，故其可用以预断(prognosis)一个体是否具有罹患胆管癌的风险。因此，本发明提供一种用以分析一生物样本的整合式微流体芯片，以及一种基于该整合式微流体芯片所产生的分析结果，来预断一个体是否罹患或具有罹患胆管癌的风险的方法。
据此，本发明的第一方面是关于一种磺化八糖的新颖用途，其可作为一种用以鉴定一生物样本中的胆管癌细胞的生物标记。该磺化八糖具有式(I)的结构：
其中R1是-NHAc或-NHSO3M；R2与R3各自为H或-SO3M；且M是一单价阳离子，其选自由钠离子、钾离子、锂离子及铵离子所组成的群组。
依据本发明的一较佳实施例，在该式(I)中，R1是–NHSO3Na；且R2与R3各自为-SO3Na。
依据本发明的另一较佳实施例，在该式(I)中，R1是-NHSO3Na，R2与R3各自为H。
本发明的第二方面是关于一种用以检测一生物样本中的胆管癌细胞的方法。该方法包括一步骤，其中该步骤是使一生物样本与一预先涂布上述式(I)的磺化八糖或其药学上可接受的盐的磁珠接触，其中该生物样本与该磁珠的间的结合表示该生物样本中具有胆管癌细胞。
依据本发明的实施方式，式(I)的磺化八糖还与一生物素(biotin)连接。
依据本发明的一较佳实施方式，该磁珠是以式(I)的磺化八糖预先涂布，其中R1是-NHSO3Na，R2与R3各自为H。
依据本发明的其他实施方式，该磁珠是以式(I)的磺化八糖预先涂布，其中R1是-NHSO3Na；且R2与R3各自为-SO3Na。
依据本发明的实施方式，该生物样本可为血液、血浆、血清、尿液、痰液、唾液、组织样本、活体组织切片或组织溶解产物。
本发明的第三方面是关于一种用以分析一生物样本的微流体芯片，例如确认该生物样本中是否具有癌症。该微流体芯片包含：
复数个洗涤缓冲液腔(wash buffer chambers)，其分别用以滞留(hold)其中的洗涤缓冲液；
复数个捕捉腔(capture chambers)，其分别用以将一癌细胞捕捉至一预先涂布癌细胞生物标记的磁珠上；
一个废液腔(waste chamber)，其用以滞留自该复数个捕捉腔中洗出的未捕捉的癌细胞；以及
复数个微通道(microchannels)，其用以连接该复数个洗涤缓冲液腔、该复数个捕捉腔与该废液腔。
依据本发明的实施方式，该微流体芯片是由一玻璃基板和至少一聚二甲基硅氧烷(polydimethylsiloxane，PDMS)层所制成。
依据本发明的一较佳实施方式，该微流体芯片的结构包含六个洗涤缓冲液腔、六个捕捉腔和一个废液腔，其分别与彼此连接并以微通道相通。
依据本发明的实施方式，各该捕捉腔是用以分离出一液体样本中与磁珠结合的癌细胞。
依据本发明的实施方式，该磁珠是以式(I)的磺化八糖预先涂布，
其中，
R1是-NHAc或-NHSO3M；R2与R3各自为H或-SO3M；且M是一单价阳离子，其选自由钠离子、钾离子、锂离子及铵离子所组成的群组。
依据本发明的实施方式，该式(I)的磺化八糖还与一生物素连接。
依据本发明的实施方式，在该式(I)的磺化八糖中，R1是-NHSO3Na；且R2与R3各自为-SO3Na。
依据本发明的实施方式，在该式(I)的磺化八糖中，R1是-NHSO3Na，R2与R3各自为H。
本发明的第四方面是关于一种用以分析一生物样本的试剂盒。该试剂盒包括：本发明中微流体芯片、预先涂布一生物标记的磁珠、至少一试剂，其用以分析利用本发明微流体芯片所得的生物样本；以及一用以指示使用者如何使用该试剂盒的使用说明。
依据本发明的特定实施方式，该生物标记为上述式(I)的磺化八糖，其中R1是-NHSO3Na；且R2与R3各自为-SO3Na。
依据本发明的另一实施方式，该生物标记为上述式(I)的磺化八糖，其中R1是-NHSO3Na，R2与R3各自为H。
本发明的一或多个实施方式的细节将在以下的叙述中呈现。根据详细说明和申请专利范围，本发明的其他特征和优点将更显而易见。
当可理解，前面的一般性描述和以下的详细描述都是通过实施例，并且旨在对本发明的提供进一步说明。
附图说明
本发明或申请说明书中包含至少一张彩色附图。可向专利局申请并缴交必要费用后，取得此一包含至少一张彩色附图的本发明或申请说明书副本。
以下随附图式包含在说明书中并构成说明书的一部分，阐述了本发明不同方面的实施例***、方法与例示范性实施方式。在参阅以下的详细说明及附图后，本发明将更明显易懂，其中：
图1：微流体芯片的示意图。(a)微流体芯片的示意图，其自底部到顶部包括一玻璃基板、一PDMS薄层及一PDMS厚层。(b)一示意图，其说明刻在PDMS层中的洗涤缓冲液腔、捕捉腔、废液腔及微通道。
图2：图片阐述以HS-1涂布的磁珠来捕捉Huh-28细胞。(a)未捕捉的Huh-28细胞；(b)在作用30分钟后，以HS-1涂布的磁珠来捕捉Huh-28细胞；以及(c)自磁珠与细胞的复合物中所收集的上清液。所有影像均为100×放大倍数。
具体实施例方式
下文提供了详细说明及附图是为使本发明实施例的叙述更加详尽与完备，而非代表用以理解或运用本发明实施例的唯一形式。
1.定义
除非另有指明，所述“个体”(subject)或“病患”(patient)在本发明内可互换使用，是指任一种动物。该动物可以是一人类或非人类个体。该个体可以是人类；但也可以是一需要兽医治疗的哺乳动物，例如家畜或比赛用动物，农用动物和实验动物(例如大鼠、小鼠、豚鼠、灵长类等)。通常该动物是一非人类的哺乳类动物，例如非人类的灵长类动物。非人类的灵长类动物包括黑猩猩、食蟹猴、蜘蛛猴和猕猴(例如恒河猴或黑猩猩(Pan))。家畜或比赛用动物包括牛、马、猪、羊、鹿、野牛、水牛、貂、猫科动物(例如家猫)、犬科动物(例如狗、狼和狐狸)、禽类(例如鸡、火鸡和鸵鸟)和鱼(例如鳟鱼、鲶鱼和鲑鱼)。
在此所述“接触”(contacting)在本文中是与细胞(例如一生物样本中的细胞)相关，且是指任何将试剂递送或“施予”(administration)至细胞或生物样本的方式，其中将所述试剂(例如本发明的磺化八糖或预先涂布本发明的磺化八糖的磁珠)与一或多个细胞接触，其量足以达到彼此间的亲和性结合。
尽管在本发明概括的范围所列举的数值范围与参数设定为近似值，于具体实施例中所提出的数值则尽可能地精确。然而，任何数值与生俱有因各别测试性测量产生的标准偏差而产生的某些误差。同样地，如同文内所使用，所谓的“约”(about)通常表示给定值或范围的10％、5％、1％或0.5％。或者，在本领域技术人员的观点，所述“约”意指在平均值的可接受的标准误差内。除了操作或工作实例，或除非另有特别说明，所有数值范围、数量、数值和百分比，例如材料数量、持续时间、温度、操作条件、数量比值，和其他本文内公开所有相似术语，应理解为在所有情况下均由“约”修饰。至少，每个数值参数应根据报告的有效数字的数量与通过普通的舍入技术来解释。
除非本说明书另有定义，本说明书所用的单数名词涵盖该名词的复数型。
2.本发明的磺化八糖
本发明一方面涉及关于发现特定的磺化八糖可作为方法和/或试剂盒中的生物标记，用以鉴定和/或检测一生物样本中的癌细胞。例示性的磺化八糖于本文中描述。
在一方面中，本发明是关于式(I)的磺化八糖：
其中R1是-NHAc或-NHSO3M；R2与R3各自为H或-SO3M；且M是一单价阳离子，其选自由钠离子、钾离子、锂离子及铵离子所组成的群组。
在本发明的某些实施方式中，R1是-NHSO3Na；且R2与R3各自为-SO3Na。
在本发明的进一步实施方式中，R1是-NHSO3Na，且R2与R3各自为H。
可依据操作实施例中所描述的方法来制备本发明的磺化八糖。所有本发明化合物的立体异构物，例如这些因式(I)的化合物的R取代基上的不对称碳原子而存在的立体异构物，包含镜像和非镜像形式，都视为涵盖在本发明的范围内。本发明化合物中的各立体异构物可以是，举例来说，实质上不含其它异构物，或者可以是混合物，举例来说，是外消旋物、或与其他所有或其他特定异构物的混合。此外，本发明的手性中心可具有以IUPAC 1974命名法定义的S或R构型。
3.使用方法
式(I)的磺化八糖可与癌细胞表面结合，特别是源自于胆管的癌症。因此，本发明涵盖一种用以鉴定或检测一生物样本中的胆管癌细胞的方法。
本发明方法包括使一个体的生物样本与一预先涂布式(I)磺化八糖的磁珠接触，其中该磁珠与该生物样本的间的结合表示该生物样本中具有胆管癌细胞。
依据本发明的实施方式，预先涂布式(I)磺化八糖的磁珠还与一生物素连接。
依据本发明的较佳实施方式，在该式(I)的磺化八糖中，R1是-NHSO3Na，R2与R3各自为H。
依据本发明的其他实施方式，在该式(I)的磺化八糖中，R1是-NHSO3Na；且R2与R3各自为-SO3Na。
适用于本发明方法的生物样本实例包括，但不限于，血液、血浆、血清、尿液、痰液、唾液、组织样本、活体组织切片及组织溶解产物。在一较佳实施方式中，该生物样本为血液。
本发明因此提供一种用以预断一个体是否罹患胆管癌的方法。该预断为对一个体的样本来进行，其可以是以下任一种：血液、血浆、血清、尿液、痰液、唾液、组织样本、活体组织切片及组织溶解产物。该方法包含：将组织样本与预先涂布式(I)的磺化八糖的磁珠作用足够时间，其中，若观察到该组织样本与该预先涂布式(I)的磺化八糖的磁珠发生结合，则该个体罹患胆管癌。
依据本发明的特定实施方式，组织样本可以是血液、血浆、血清、尿液、痰液、唾液、组织样本、活体组织切片或组织溶解产物。在一较佳实施方式中，该组织样本为血液。
依据本发明的较佳实施方式，该磁珠是以式(I)的磺化八糖预先涂布，其中R1是-NHSO3Na，R2与R3各自为H。
依据本发明的其他实施方式，该磁珠是以式(I)的磺化八糖预先涂布，其中R1是-NHSO3Na；且R2与R3各自为-SO3Na。
4.微流体芯片
在特定实施方式中，本发明是关于一种集成微流体芯片，其可加速检测和/或分离一生物样本中的癌细胞。
参照图1，其为本发明的微流体芯片100的示意图，其由一玻璃基板与至少一聚二甲基硅氧烷(PDMS)层所制成。如图1(a)所示，微流体芯片100自底部到顶部包含一玻璃基板110、一第一PDMS层120，与一第二PDMS层130，其中该第一与该第二PDMS层120、130分别作为一液体通道层与一空气通道层。较佳地，该第一PDMS层120的厚度较第二PDMS层130薄。可通过本领域已知的计算机数值控制(computer numerical control，CNC)机械加工工艺(machining process)(例如EGX-400,Roland Inc.,Japan)来将微结构(microstructures)(例如流体腔(fluid chambers)与流体导管(fluid conducts)(或流体通道(fluidchannels)))建构或蚀刻在该第一与第二PDMS层120、130上。
图1(b)为在第二PDMS层130上建构流体腔和/或流体导管的代表示意图。如图所示，在PDMS层130上建构6个洗涤缓冲液腔102(a到f)、6个捕捉腔104(a到f)、1个废液腔106，与复数个微通道108。为求简洁，在图1(b)中，仅标示出6个洗涤缓冲液腔中的一个(即102)与6个捕捉腔中的一个(104)。这些腔室(chambers)以环状排列，并将废液腔106设置于PDMS层的中心，6个捕捉腔104(a到f)蚀刻在废液腔106的周围，且各捕捉腔通过复数个微通道108与相邻的捕捉腔及废液腔连接。接着，将6个洗涤缓冲液腔102(a到f)蚀刻在6个捕捉腔104(a到f)的周围，且各洗涤缓冲液腔通过复数个微通道108与相邻的洗涤缓冲液腔及捕捉腔连接。通过这样的排列，各洗涤缓冲液腔102(a到f)、捕捉腔104(a到f)与废液腔106通过复数个微通道108可达到流体连通。可将腔室与微通道以相同的方式建构在第一PDMS层120上。
操作时，首先将合适量的液体样本(例如血液或血浆)注入各捕捉腔104(a到f)，接着将预先涂布生物标记(例如本发明式(I)的磺化八糖)的磁珠加入各捕捉腔104(a到f)中，并使各捕捉腔104(a到f)中的混合物反应足够时间(例如至少15分钟)，使癌细胞可通过亲和力结合而与磁珠上的生物标记结合。接着以磁力收集其上带有期望的癌细胞的磁珠，而这些在生物样本中未结合的癌细胞与其他成分则以装载于洗涤缓冲液腔102(a到f)的洗涤缓冲液洗出并收集在废液腔106中。
根据本发明的较佳实施例，该微流体芯片由6个洗涤缓冲液腔与6个捕捉腔所组成。因此，此微流体芯片可在六种不同条件下鉴定和/或捕捉所需的癌细胞，例如分别使用六种其上涂布不同类型的生物标记的磁珠，或是使用一种生物标记但六种不同结合条件(例如酸碱值、离子强度等)。通过这样的方式，可加速检测流体样本中癌细胞，其中检测相对更完整，因为可采用更广范围的生物标记和/或条件。
在本发明的一较佳实施例中，将预先涂布本发明式(I)的磺化八糖的磁珠各自装载在微流体芯片的捕捉腔中，并以预先涂布磺化八糖HS1与HS12的磁珠来辨识胆管癌细胞。
此外或可任选性地，各磁珠还可与一生物素(biotin)连接以放大检测讯号。
5.试剂盒
本发明另一方面是关于一种用以辨识和/或检测一生物样本中的癌细胞的试剂盒。该试剂盒至少包括，本发明的微流体芯片、预先涂布生物标记的磁珠、至少一试剂，其用以分析利用本发明微流体芯片所得的生物样本，以及一用以指示使用者如何使用该试剂盒的使用说明。
根据本发明的较佳实施例，该试剂盒至少包含三个容器，与一份用以指示使用者如何使用该试剂盒的使用说明。第一容器可于其中储存本发明的微流体芯片。第二容器可于其中容纳预先涂布磺化八糖HS1的磁珠、磺化八糖HS12的磁珠、或其混合物。第三容器可于其中储存分析时所必需的缓冲溶液，例如用以清洗未与磁珠结合的癌细胞。使用说明可以是小册子、录音带、CD、VCD或DVD等形式。
下文针对了本发明的实施方面与具体实施例提出了说明性的描述；以利本发明所属技术领域中具有通常知识者实作本发明，且不应将这些实验例视为对本发明范围的限制。尽管用于这些具体实施例的方法为那些典型常用的方法，其他为本领域技术者所熟知的程序、方法、技术也有可能替代使用。
实施例
材料与方法
细胞培养：将HEK293T(人类胚胎肾细胞)与COS-1(猴肾细胞)细胞培养在混有10％已热去活的胎牛血清的杜氏改良式依格尔培养基(Dulbecco’s modified eagle’smedium，DMEM)中。将Neuro2a(源自小鼠神经母细胞瘤)细胞培养在混有10％胎牛血清的RPMI-1640培养基中。
将SNU478、HuCCT1、Huh28、BxPC3与HCT8细胞分别培养于含有盘尼西林(100U/毫升)与链霉素(100微克/毫升)混合液(Pen Strep,
Invitrogen Co.,USA)、10％胎牛血清(FBS,
Invitrogen Co.,USA)的RPMI-1640培养基(
InvitrogenCo.,USA)中。将KKU100、MMNK1与HepG2细胞则分别培养在含有盘尼西林(100U/毫升)与链霉素(100微克/毫升)混合液，10％胎牛血清(FBS,
)的DMEM培养基内。所有的细胞皆培养在37℃、5％CO2的环境中。
制备预先涂布磺化八糖的磁珠：将磁珠表面分别预先涂布三种不同浓度(1、10、100微体积摩尔浓度)的本发明磺化八糖(例如HS1、HS12等)。简单来说，首先将磺化八糖与链亲和素T1磁珠(
MyOneTM Streptavidin T1)(～7-10×109个/毫升，
微米，Invitrogen Co.,USA)以体积比1：10的比例培养30分钟。接着以磁铁吸附5分钟收集磁珠，之后去除上清液并以1毫升的去离子水(deionized，DI)清洗三次。最后，再将涂布完成的磁珠悬浮在DI水中，其体积与一开始自
瓶中取出的体积相同。
建构微流体芯片：为能使磁珠有效地捕捉癌细胞，依据蔡(Tsai W.C.)等人所述方法(公开于2017年4月9日至4月12日于美国洛杉矶市举办的第12届IEEE世界年会“以八糖磺化乙酰肝素来检测胆管癌细胞的微流***”文章)来建构集成微流体芯片。简言之，此集成微流体芯片是以两层聚二甲基硅氧烷(polydimethylsiloxane,PDMS)层与一玻璃基板所制成；二PDMS层中的其中一层比另一层厚，此PDMS厚层是作为空气通道层，而PDMS薄层则是作液体通道层，最后，玻璃基板则用于密封PDMS层。
通过计算机数值控制(computer-numerical-control,CNC)机械加工工艺(EGX-400,Roland Inc.,Japan)搭配0.5毫米钻头，来将空气与液体通道层的母模型蚀刻在聚甲基丙烯酸甲酯(polymethylmethacrylate，PMMA)上的微结构中。接着，进行聚二甲基硅氧烷(PDMS)铸造与翻模工艺以获得母模型的逆向结构(inverse structures)。通过混合重量比例为1:10的固化剂与PDMS预聚合物(Sylgard 184A/B,Sil-More Industrial Ltd.,USA)，并置于真空中以去除所有的气泡，来制备聚二甲基硅氧烷(PDMS)。在去除气泡40分钟后，以手动填充方式将PDMS混合物填入母模型并于70℃中固化2小时。接着，以机械式地从母模型剥离两个固化的PDMS层，再以等离子体氧化方式将该厚的与薄的PDMS层互相黏合。最后再以同样的等离子体氧化方式将组装好的PDMS层与玻璃基板互相黏合，而形成微流体芯片。
以微流体芯片捕捉癌细胞
利用上述建构的微流体芯片来捕捉癌细胞。简言之，分别将2×105个不同种类的癌细胞株悬浮于100微升的1倍磷酸盐缓冲溶液(phosphate buffered saline，PBS)中，并注入六个捕捉腔中。将10微升的不同浓度的磺化八糖磁珠以手动方式分注到捕捉腔中，并以微型泵温和地与细胞混合15分钟。在作用后，预先涂布本发明中式(I)磺化八糖的磁珠会辨认并结合细胞。接着，以磁力收集被捕捉的细胞，并用1倍磷酸盐缓冲溶液洗涤。在分离后，以100微升的1倍磷酸盐缓冲溶液来悬浮磁珠与细胞，再将样本加载到血球计数器中以计算捕捉率。
统计分析
此处并无统计学方法用于预先确定实验的样本量。以平均值±标准误(s.e.m.)或标准偏差(s.d.)来表示(于各个图式说明中指出)分析数据结果。以T检定来比较两组数据以分析数据组。在二组数据以上的情形，则使用单因子变异数分析法(one-way ANOVA)，搭配Duncan’s或Tukey检定以校正多重比较。所有检定均以双面方式进行。P值等于或小于0.05的实验结果是具有显著差异。使用Excel 2013(Microsoft software)来计算平均值、标准偏差、标准误差与统计分析。在本研究中没有使用包含或排除数据的判断标准。
实施例1化学合成本发明磺化八糖
可依据流程I和流程II所述步骤来分别合成本发明两组磺化八糖，分别为HS1～HS8与HS9～HS16。大体上，是使用双糖与四糖建构组元(building blocks)搭配硫代甲苯官能基(thiotolyl)作为离去基，并以收敛聚合方式形成糖骨架。根据最近用于肝素和肝素合成的模式(Hu et al.,2011Nat.Chem.3,557-563；Zulueta et al.,2012J.Am.Chem.Soc.134,8988-8995)来选用保护基。对于完全保护的骨架，则以发散方式(divergent manner)(Hu et al.,2011Nat.Chem.3,557-563；Zulueta et al.,2012J.Am.Chem.Soc.134,8988-8995)来进行官能基转换，以提供带有一氨基戊基糖苷配基基团(aminopentyl aglycon moiety)的不同磺化终产物。
流程I
流程II
以下提供HS1至HS16的化学数据。
5-氨基戊基(2-乙酰胺基-2-脱氧-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆基(idopyranosiduronyl)-(1→4)-(2-乙酰胺基-2-脱氧-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆基-(1→4)-(2-乙酰胺基-2-脱氧-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆基-(1→4)-(2-乙酰胺基-2-脱氧-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆酸酯(idopyranosiduronate)(HS1)。1H NMR(600MHz,D2O):δ5.12–5.06(m,4H),4.84–4.82(m,H),4.79–4.76(m,2H),4.67–4.63(m,3H),4.42(d,J＝2.8Hz,1H),4.02–3.93(m,4H),3.90–3.83(m,5H),3.83–3.71(m,15H),3.71–3.62(m,8H),3.62–3.53(m,4H),3.53–3.49(m,1H),3.38(t,J＝9.5Hz,1H),2.90(t,J＝7.6Hz,2H),1.94(s,3H,Ac),1.93(s,6H,Ac×2),1.92(s,3H,Ac),1.63–1.54(m,4H,CH2连接子),1.40-1.32(m,2H,CH2连接子)；13C NMR(150MHz,D2O):δ175.0(C),174.9(C),174.8(C),174.3(C),174.28(C),174.2(C),101.6(CH),100.7(CH),94.4(CH),94.3(CH),76.6(CH),76.5(CH),76.4(CH),74.5(CH),74.4(CH),74.3(CH),73.9(CH),71.9(CH),71.1(CH),71.05(CH),71.0(CH),69.9(CH),69.8(CH),69.7(CH),69.6(CH),69.4(CH),69.3(CH),68.8(CH),68.6(CH),68.0(CH2),60.1(CH2),60.0(CH2),53.6(CH),53.5(CH),39.3(CH2),28.0(CH2),26.2(CH2),22.2(CH2),21.8(CH3)；HRMS(ESI):m/z计算为C61H95N5O45([M–2H]2–):808.7650,实测值:808.7641。
5-氨基戊基(2-乙酰胺基-2-脱氧-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆基-(1→4)-(2-乙酰胺基-2-脱氧-α-D-葡萄吡喃糖基)-(1→4)-β-D-葡萄吡喃环草隆基(glucopyranosiduronyl)-(1→4)-(2-乙酰胺基-2-脱氧-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆基-(1→4)-(2-乙酰胺基-2-脱氧-α-D-葡萄吡喃糖基)-(1→4)-β-D-葡萄吡喃环草隆酸酯(glucopyranosiduronate)(HS9)。1H NMR(600MHz,D2O):δ5.40(d,J＝3.8Hz,1H),5.38(d,J＝3.8Hz,1H),5.20(d,J＝3.7Hz,1H),5.19(d,J＝3.7Hz,1H),4.94(d,J＝3.7Hz,1H),4.93(d,J＝3.9Hz,1H),4.75(d,J＝3.1Hz,1H),4.74(d,J＝2.9Hz,1H),4.51(d,J＝7.9Hz,1H),4.46(d,J＝8.0Hz,1H),4.10(t,J＝6.8Hz,1H),4.08(t,J＝6.6Hz,1H),3.97–3.95(m,1H),3.95–3.91(m,5H),3.90–3.87(m,5H),3.86–3.83(m,7H),3.83–3.79(m,4H),3.79–3.77(m,2H),3.77–3.76(m,2H),3.76–3.74(m,2H),3.74–3.71(m,3H),3.71–3.69(m,4H),3.69–3.66(m,1H),3.49(t,J＝9.4Hz,1H),3.38(t,J＝8.6Hz,1H),3.31(t,J＝8.6Hz,1H),3.01(t,J＝7.5Hz,1H),2.06(s,6H,Ac×2),2.04(s,3H,Ac),2.03(s,3H,Ac),1.73-1.65(m,4H),1.52–1.43(m,2H)；13C NMR(150MHz,D2O):δ176.0(C),175.9(C),175.82(C),175.77(C),175.3(C),175.24(C),175.20(C),103.3(CH),103.2(CH),102.8(C),102.7(CH),97.95(CH),95.4(CH),95.3(CH),79.1(CH),78.1(CH),77.91(CH),77.87(CH),77.7(CH),77.5(CH),77.3(CH),77.19(CH),77.17(CH),75.6(CH),75.3(CH),74.5(CH),74.4(CH),72.9(CH),72.1(CH),71.7(CH),71.03(CH2),71.00(CH),70.95(CH),70.9(CH),70.8(CH),70.7(CH),70.6(CH),70.5(CH),70.4(CH),61.2(CH2),60.7(CH2),60.6(CH2),60.4(CH2),54.8(CH),54.5(CH),54.1(CH),40.4(CH2),29.1(CH2),27.3(CH2),22.9(CH2),22.9(CH3)；HRMS(ESI):m/z计算为C61H94N5Na5O45([M+5Na–3H]2+):865.7355,实测值:865.7357。
5-氨基戊基(2-脱氧-2-磺氨基(sulfamido)-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆基-(1→4)-(2-脱氧-2-磺氨基-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆基-(1→4)-(2-脱氧-2-磺氨基-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆基-(1→4)-(2-脱氧-2-磺氨基-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆酸酯(HS2)。1H NMR(600MHz,D2O):δ5.41–5.35(m,4H),4.98–4.94(m,2H),4.92–4.89(m,2H),4.51(d,J＝1.9Hz,1H),4.16–4.02(m,8H),3.92–3.59(m,27H),3.47(t,J＝9.4Hz,1H),3.28–3.19(m,4H),3.00(t,J＝7.4Hz,2H),1.72–1.63(m,2H),1.48–1.43(m,2H)；13C NMR(150MHz,D2O):δ175.2(C),101.6(CH),100.7(CH),95.64(CH),95.6(CH),95.5(CH),95.4(CH),77.1(CH),77.0(CH),76.9(CH),74.9(CH),74.8(CH),74.7(CH),71.7(CH),71.3(CH),71.0(CH),70.9(CH),69.8(CH),69.73(CH),69.7(CH),69.3(CH),69.1(CH),69.0(CH),68.8(CH),68.5(CH),68.4(CH),68.2(CH),68.1(CH2),60.2(CH2),59.7(CH2),58.0(CH),57.8(CH),39.4(CH2),28.0(CH2),26.2(CH2),22.2(CH2)；HRMS(ESI):m/z计算为C53H86N5O53S4Na3([M+3Na–6H]3–):611.4177,实测值:611.4167。
5-氨基戊基(2-乙酰胺基-2-脱氧-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-α-L-艾杜吡喃环草隆基)-(1→4)-(2-乙酰胺基-2-脱氧-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-α-L-艾杜吡喃环草隆基)-(1→4)-(2-乙酰胺基-2-脱氧-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-α-L-艾杜吡喃环草隆基)-(1→4)-(2-乙酰胺基-2-脱氧-α-D-葡萄吡喃糖基)-(1→4)-2-O-磺酸根基-α-L-艾杜吡喃环草隆酸酯(HS7)。1H NMR(600MHz,D2O):δ5.17(bs,4H),5.11–5.08(m,4H),4.90(d,J＝10.1Hz,2H),4.54(d,J＝1.9Hz,1H),4.32(bs,3H),4.30-4.25(m,4H),4.21(bs,1H),4.03–3.96(m,9H),3.90–3.83(m,9H),3.82–3.76(m,4H),3.75–3.67(m,8H),3.45(t,J＝9.3Hz,1H),2.99(t,J＝7.5Hz,2H),2.05(s,6H,Ac×2),2.04(s,6H,Ac×2),1.70–1.61(m,4H,CH2连接子),1.50–1.40(m,2H,CH2连接子)；13C NMR(150MHz,D2O):δ175.8(C),175.2(C),174.8(C),174.7(C),99.3(CH),99.2(CH),98.6(CH),93.5(CH),93.4(CH),93.3(CH),77.2(CH),74.1(CH),73.3(CH),73.2(CH),73.1(CH),71.9(CH),71.3(CH),71.2(CH),70.7(CH),70.4(CH),70.2(CH),69.9(CH),69.8(CH),69.7(CH),68.1(CH2),67.4(CH),67.3(CH),67.2(CH),64.0(CH),63.4(CH),63.3(CH),63.1(CH),60.2(CH2),59.7(CH2),39.4(CH2),27.8(CH2),26.2(CH2),22.24(CH3),22.2(CH3),22.1(CH3)；HRMS(ESI):m/z计算为C61H94N5O57S4([M–3H]3–)645.4498,实测值645.4489。
5-氨基戊基(2-脱氧-2-磺氨基-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆基-(1→4)-(2-脱氧-2-磺氨基-α-D-葡萄吡喃糖基)-(1→4)-β-D-葡萄吡喃环草隆基-(1→4)-(2-脱氧-2-磺氨基-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆基-(1→4)-(2-脱氧-2-磺氨基-α-D-葡萄吡喃糖基)-(1→4)-β-D-葡萄吡喃环草隆酸酯(HS10)。1H NMR(600MHz,D2O):δ5.62(d,J＝3.7Hz,1H),5.61(d,J＝3.6Hz,1H),5.41(d,J＝3.4Hz,1H),5.39(d,J＝3.4Hz,1H),4.98(bs,1H),4.96(d,J＝2.8Hz,1H),4.55(d,J＝7.9Hz,1H),4.50(d,J＝8.0Hz,1H),4.14–4.07(m,4H),3.94–3.88(m,4H),3.88–3.84(m,6H),3.84–3.81(m,7H),3.81–3.78(m,4H),3.76–3.74(m,3H),3.74–3.70(m,4H),3.68–3.63(m,3H),3.48(t,J＝9.5Hz,1H),3.43(t,J＝8.6Hz,1H),3.36(t,J＝8.6Hz,1H),3.31–3.25(m,3H),3.23(dd,J＝10.3,3.6Hz,1H),3.02(t,J＝7.5Hz,2H),1.75–1.65(m,4H),1.53–1.43(m,2H)；13C NMR(150MHz,D2O):δ176.3(C),176.1(C),176.0(C),103.3(CH),103.2(CH),102.8(CH),102.7(CH),98.5(CH),98.4(CH),96.5(CH),96.4(CH),78.9(CH),78.3(CH),78.11(CH),78.06(CH),77.94(CH),77.89(CH),77.6(CH),77.5(CH),76.0(CH),75.9(CH),73.81(CH),73.76(CH),72.7(CH),72.4(CH),71.94(CH),71.91(C),71.6(CH),71.1(CH2),70.9(CH),70.84(CH),70.80(CH),70.5(CH),70.33(CH),70.28(CH),70.0(CH),69.6(CH),61.4(CH2),60.81(CH2),60.79(CH2),60.5(CH2),59.3(CH),59.0(CH),58.6(CH),40.5(CH2),29.2(CH2),27.3(CH2),23.0(CH2)；HRMS(ESI):m/z计算为C53H81N5Na8O53S4([M+3Na–6H]3–):611.4177,实测值:611.4176；计算为C53H81N5Na8O53S4([M+4Na–7H]3–):618.7449,实测值:618.7449。
5-氨基戊基(2-乙酰胺基-2-脱氧-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-α-L-艾杜吡喃环草隆基)-(1→4)-(2-乙酰胺基-2-脱氧-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-β-D-葡萄吡喃环草隆基-(1→4)-(2-乙酰胺基-2-脱氧-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-α-L-艾杜吡喃环草隆基)-(1→4)-(2-乙酰胺基-2-脱氧-α-D-葡萄吡喃糖基)-(1→4)-2-O-磺酸根基-β-D-葡萄吡喃环草隆酸酯(HS13)。1H NMR(600MHz,D2O):δ5.39(d,J＝3.6Hz,1H),5.38(d,J＝3.4Hz,1H),5.23–5.20(m,2H),5.14–5.12(m,2H),4.92(d,J＝1.6Hz,1H),4.89(s,1H),4.73(d,J＝7.7Hz,1H),4.63(d,J＝7.9Hz,1H),4.35(s,1H),4.34(s,1H),4.32(s,1H),4.28(s,1H),4.16(t,J＝8.3Hz,1H),4.10(t,J＝8.7Hz,1H),4.06–4.00(m,4H),4.00–3.97(m,2H),3.95–3.90(m,5H),3.88–3.85(m,10H),3.84–3.80(m,6H),3.78–3.66(m,6H),3.48(t,J＝9.3Hz,1H),3.03(t,J＝7.4Hz,2H),2.09(bs,3H,Ac),2.08(bs,3H,Ac),2.07(bs,3H,Ac),2.06(bs,3H,Ac),1.74–1.65(m,4H,CH2连接子),1.56–1.48(m,2H,CH2连接子)；13C NMR(150MHz,D2O):δ176.8(C),176.4(C),175.9(C),175.8(C),175.7(C),175.6(C),175.4(C),175.3(C),101.7(CH),101.6(CH),100.4(CH),100.3(CH),98.3(CH),98.2(CH),94.70(CH),94.65(CH),81.6(CH),81.3(CH),80.7(CH),78.4(CH),77.7(CH),77.6(CH),77.4(CH),77.2(CH),76.6(CH),76.4(CH),74.8(CH),74.4(CH),73.0(CH),72.4(CH),72.34(CH),72.30(CH),71.8(CH),71.5(CH),71.1(CH2),70.9(CH),70.6(CH),70.5(CH),68.6(CH),68.5(CH),65.1(CH),64.4(CH),61.4(CH2),60.7(CH2),60.5(CH2),55.1(CH),54.5(CH),54.1(CH),40.5(CH2),29.0(CH2),27.3(CH2),23.3(CH3),23.0(CH2),22.9(CH3)；HRMS(ESI):m/z计算为C61H91N5O57S4Na8([M+8Na–6H]2+):1058.6221,实测值:1058.6226。
5-氨基戊基(2-乙酰胺基-2-脱氧-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆基-(1→4)-(2-乙酰胺基-2-脱氧-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-β-D-葡萄吡喃环草隆基-(1→4)-(2-乙酰胺基-2-脱氧-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆基-(1→4)-(2-乙酰胺基-2-脱氧-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-β-D-葡萄吡喃环草隆酸酯(HS15)。1H NMR(600MHz,D2O):δ5.43–5.38(m,2H),5.21–5.18(m,2H),5.01–4.99(m,2H),4.62–4.58(m,2H),4.49–4.46(m,2H),4.39–4.36(m,4H),4.25–4.22(m,3H),4.12–4.10(m,3H),4.02–3.98(m,5H),3.99–3.96(m,2H),3.93–3.91(m,2H),3.85–3.82(m,3H),3.80–3.73(m,14H),3.61–3.57(m,2H),3.39–3.36(m,2H),3.35–3.31(m,1H),3.04–3.00(m,2H),2.07(bs,3H,Ac),2.06(bs,3H,Ac),2.04(bs,3H,Ac),2.03(bs,3H,Ac),1.71–1.67(m,4H,CH2连接子),1.52–1.46(m,2H,CH2连接子)；13C NMR(150MHz,D2O):δ176.1(C),176.02(C),175.99(C),175.42(C),175.37(C),175.32(C),175.28(C),103.3(CH),103.09(CH),103.06(CH),103.03(CH),103.0(CH),102.9(CH),98.1(CH),95.5(CH),83.1(CH),80.9(CH),78.4(CH),77.9(CH),77.8(CH),77.7(CH),77.6(CH),77.5(CH),77.3(CH),76.2(CH),75.2(CH),74.62(CH),74.57(CH),74.1(CH),73.0(CH),72.2(CH),71.2(CH),71.1(CH2),70.7(CH),70.6(CH),70.5(CH),70.4(CH),70.3(CH),70.2(CH),70.1(CH),70.0(CH),67.4(CH2),67.2(CH2),66.9(CH2),54.8(CH),54.5(CH),54.1(CH),40.5(CH2),29.2(CH2),27.3(CH2),23.0(CH3)。
5-氨基戊基(2-乙酰胺基-2-脱氧-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆基-(1→4)-(2-乙酰胺基-2-脱氧-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆基-(1→4)-(2-乙酰胺基-2-脱氧-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆基-(1→4)-(2-乙酰胺基-2-脱氧-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆酸酯(HS3)。1H NMR(600MHz,D2O):δ5.23–5.10(m,7H),5.03–4.93(m,3H),4.33–4.20(m,12H),4.16–3.96(m,11H),3.95–3.84(m,6H),3.83–3.66(m,10H),3.63–3.58(m,2H),3.10–3.00(m,1H),2.08(bs,6H,Ac),2.04(bs,3H,Ac),2.03(bs,3H,Ac),1.80–1.65(m,4H,CH2连接子),1.52–1.46(m,2H,CH2连接子)；13C NMR(150MHz,D2O):δ175.5(C),175.3(C),175.2(C),175.0(C),174.8(C),174.4(C),102.0(CH),101.8(CH),100.8(CH),99.2(CH),94.4(CH),93.7(CH),74.0(CH),71.2(CH),71.0(CH),70.7(CH),70.1(CH),70.0(CH),69.8(CH),69.4(CH),69.3(CH),69.1(CH),69.0(CH),68.6(CH),68.1(CH),66.4(CH),66.2(CH),66.1(CH2),53.6(CH),53.4(CH),53.2(CH),53.1(CH),39.4(CH2),28.0(CH2),26.1(CH2),22.2(CH3)；HRMS(MALDI):m/z计算为C61H94N5O57S4([M–3H]3–):645.4498,实测值:645.4493。
5-氨基戊基(2-乙酰胺基-2-脱氧-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-α-L-艾杜吡喃环草隆基)-(1→4)-(2-乙酰胺基-2-脱氧-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-α-L-艾杜吡喃环草隆基)-(1→4)-(2-乙酰胺基-2-脱氧-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-α-L-艾杜吡喃环草隆基)-(1→4)-(2-乙酰胺基-2-脱氧-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-2-O-磺酸根基-α-L-艾杜吡喃环草隆酸酯(HS5)。1H NMR(600MHz,D2O):δ5.24–2.18(m 5H),5.18–5.08(m,5H),5.04–4.89(m,2H),4.35–4.20(m,13H),4.15–3.93(m,9H),3.94–3.79(m,4H),3.79–3.59(m,10H),3.56(t,J＝9.7Hz,1H),3.00(t,J＝7.2Hz,2H),2.08(s,3H,Ac),2.06(s,3H,Ac),2.01(s,6H,Ac×2),1.73–1.59(m,4H),1.52–1.38(m,2H)；13C NMR(150MHz,D2O):δ174.8(C),174.4(C),174.3(C),172.7(C),101.8(CH),100.9(CH),99.1(CH),99.0(CH),98.5(CH),95.3(CH),94.9(CH),76.3(CH),76.2(CH),73.7(CH),73.6(CH),71.0(CH),70.8(CH),70.6(CH),70.56(CH),69.7(CH),69.6(CH),68.9(CH),68.7(CH),68.6(CH2),67.4(CH),67.3(CH),66.5(CH),66.3(CH2),66.1(CH2),53.4(CH),53.3(CH),53.1(CH),39.3(CH2),27.9(CH2),27.6(CH2),26.4(CH2),22.2(CH2),22.1(CH3),21.9(CH3),21.8(CH3)；HRMS(ESI):m/z计算为C61H93N5O69S8([M–4H]4–):563.7922,实测值:563.7941。
5-氨基戊基(2-脱氧-2-磺氨基-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆基-(1→4)-(2-脱氧-2-磺氨基-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆基-(1→4)-(2-脱氧-2-磺氨基-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆基-(1→4)-(2-脱氧-2-磺氨基-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆酸酯(HS4)。1H NMR(600MHz,D2O):δ5.38–5.33(m,4H),5.02(bs,4H),4.89(bs,2H),4.51(d,J＝2.4Hz,1H),4.40–4.33(m,4H),4.25–4.17(m,4H),4.16–4.10(m,4H),4.10–4.05(m,4H),4.05–3.93(m,5H),3.88–3.83(m,1H),3.82–3.73(m,7H),3.70–3.65(m,5H),3.58(t,J＝9.7Hz,1H),3.30–3.20(m,4H),3.00(td,J＝2.1,7.5Hz,2H),1.73–1.64(m,4H,CH2连接子),1.49–1.43(m,2H,CH2连接子)；13C NMR(150MHz,D2O):δ175.4(C),175.2(C),101.9(CH),100.7(CH),95.6(CH),95.58(CH),95.5(CH),77.6(CH),77.4(CH),77.3(CH),71.2(CH),69.9(CH),69.8(CH),69.0(CH),68.9(CH),68.6(CH),68.4(CH),68.3(CH),68.0(CH2),67.8(CH),67.5(CH),66.4(CH2),66.2(CH2),57.8(CH),57.7(CH),39.4(CH2),28.0(CH2),26.2(CH2),22.2(CH2)；HRMS(ESI):m/z计算为C53H78N5O65S8Na8([M+8Na–11H]3–):754.6633,实测值:754.6641。
5-氨基戊基(2-脱氧-2-磺氨基-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-α-L-艾杜吡喃环草隆基)-(1→4)-(2-脱氧-2-磺氨基-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-β-D-葡萄吡喃环草隆基-(1→4)-(2-脱氧-2-磺氨基-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-α-L-艾杜吡喃环草隆基)-(1→4)-(2-脱氧-2-磺氨基-α-D-葡萄吡喃糖基)-(1→4)-2-O-磺酸根基-β-D-葡萄吡喃环草隆酸酯(HS14)。1H NMR(600MHz,D2O):δ5.61(d,J＝3.7Hz,1H),5.60(d,J＝3.5Hz,1H),5.35–5.32(m,2H),5.27(bs,2H),4.89–4.87(m,1H),4.72(d,J＝7.7Hz,1H),4.64(d,J＝7.8Hz,1H),4.37–4.34(m,2H),4.29–4.25(m,2H),4.18(t,J＝8.5Hz,1H),4.12(t,J＝8.6Hz,1H),4.08–4.04(m,3H),4.01–3.98(m,2H),3.90–3.84(m,12H),3.83–3.80(m,4H),3.77–3.73(m,2H),3.73–3.70(m,3H),3.70–3.65(m,5H),3.48(t,J＝9.5Hz,1H),3.28(dd,J＝10.6,3.5Hz,1H),3.27–3.22(m,3H),3.02(t,J＝7.4Hz,1H),1.72–1.63(m,4H,CH2连接子),1.57–1.46(m,2H,CH2连接子)；13C NMR(150MHz,D2O):δ176.5(C),176.2(C),175.9(C),175.6(C),101.8(CH),101.7(CH),100.2(CH),98.9(CH),98.3(CH),81.1(CH),80.7(CH),80.6(CH),78.4(CH),78.2(CH),78.1(CH),77.9(CH),77.5(CH),77.4(CH),76.6(CH),76.2(CH),75.9(CH),75.7(CH),72.7(CH),72.3(CH),72.2(CH),71.7(CH),71.10(CH2),71.08(CH),70.93(CH),70.90(CH),70.4(CH),69.5(CH),69.3(CH),69.0(CH),68.8(CH),61.4(CH2),60.8(CH2),60.4(CH2),59.4(CH),59.2(CH),58.9(CH),40.6(CH2),29.1(CH2),27.5(CH2),23.0(CH2)。
5-氨基戊基(2-脱氧-2-磺氨基-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆基-(1→4)-(2-脱氧-2-磺氨基-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-β-D-葡萄吡喃环草隆基-(1→4)-(2-脱氧-2-磺氨基-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-α-L-艾杜吡喃环草隆基-(1→4)-(2-脱氧-2-磺氨基-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-β-D-葡萄吡喃环草隆酸酯(HS16)。1H NMR(600MHz,D2O):δ5.58(bs,1H),5.38–5.32(m,2H),5.04-4.99(m,2H),4.76–4.72(m,2H),4.62–4.58(m,2H),4.46(d,J＝7.9Hz,1H),4.38–4.30(m,2H),4.21–4.17(m,3H),4.12–4.10(m,2H),4.03–4.00(m,1H),3.95–3.92(m,1H),3.88–3.83(m,2H),3.81–3.67(m,13H),3.66–3.62(m,2H),3.58–3.54(m,2H),3.53–3.49(m,1H),3.40–3.36(m,1H),3.34(d,J＝7.7Hz,1H),3.32–3.30(m,2H),3.29–3.24(m,3H),3.21(dd,J＝10.3,3.3Hz,1H),2.99(t,J＝7.4Hz,1H),1.71–1.60(m,4H,CH2连接子),1.50–1.40(m,2H,CH2连接子)；13C NMR(150MHz,D2O):δ176.51(C),176.47(C),176.1(C),176.0(C),103.2(CH),103.0(CH),102.94(CH),102.91(CH),98.7(CH),98.5(CH),96.6(CH),96.4(CH),78.7(CH),78.3(CH),78.2(CH),78.1(CH),77.8(CH),77.5(CH),77.4(CH),76.9(CH),76.7(CH),76.2(CH),75.6(CH),75.44(CH),75.40(CH),74.1(CH),73.9(CH),73.8(CH),73.0(CH),72.3(CH),71.02(CH2),70.96(CH),70.9(CH),70.7(CH),70.1(CH),70.0(CH),69.9(CH),69.8(CH),69.65(CH),69.56(CH),69.5(CH),68.8(CH),67.4(CH2),67.3(CH2),66.9(CH2),59.2(CH),58.8(CH),58.3(CH),40.5(CH2),29.2(CH2),27.3(CH2),23.0(CH2)；HRMS(ESI):m/z计算为C53H79N5O65S8Na6([M+6Na–10H]4–):554.7546,实测值:554.7562。
5-氨基戊基(2-乙酰胺基-2-脱氧-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-α-L-艾杜吡喃环草隆基)-(1→4)-(2-乙酰胺基-2-脱氧-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-β-D-葡萄吡喃环草隆基)-(1→4)-(2-乙酰胺基-2-脱氧-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-α-L-艾杜吡喃环草隆基)-(1→4)-(2-乙酰胺基-2-脱氧-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-2-O-磺酸根基-β-D-葡萄吡喃环草隆酸酯(HS11)。1H NMR(600MHz,D2O):δ5.42(s,1H),5.38(s,1H),5.23(s,2H),5.19(s,2H),4.94(s,1H),4.89(s,1H),4.64(d,J＝7.7Hz,1H),4.60(d,J＝10.6Hz,1H),4.40–4.35(m,5H),4.33(bs,1H),4.29–4.25(m,5H),4.18(t,J＝8.4Hz,1H),4.13–4.07(m,4H),4.07–4.00(m,6H),3.98–3.91(m,5H),3.89–3.81(m,9H),3.77(d,J＝9.6Hz,1H),3.75–3.70(m,2H),3.59(t,J＝9.6Hz,1H),3.04(t,J＝7.3Hz,2H),2.09(bs,6H,CH3×2),2.08(bs,3H,Ac),2.07(bs,3H,Ac),1.75–1.66(m,4H,CH2连接子),1.56–1.49(m,2H,CH2连接子)；13C NMR(150MHz,D2O):δ176.4(C),176.1(C),175.8(C),175.7(C),175.4(C),175.3(C),101.5(CH),101.0(CH),100.1(CH),100.0(CH),98.3(CH),98.1(CH),95.2(CH),94.8(CH),81.6(CH),81.1(CH),78.9(CH),77.8(CH),77.6(CH),77.3(CH),77.1(CH),76.9(CH),76.5(CH),76.4(CH),75.4(CH),74.9(CH),73.4(CH),72.3(CH),72.1(CH),71.13(CH),71.11(CH2),70.5(CH),70.4(CH),70.34(CH),70.30(CH),70.26(CH),70.1(CH),69.2(CH),68.9(CH),67.5(CH2),67.3(CH2),66.8(CH2),66.3(CH),65.2(CH),54.7(CH),54.3(CH),54.0(CH),40.5(CH2),29.0(CH2),27.3(CH2),23.3(CH3),22.97(CH2),22.95(CH3)；HRMS(ESI):m/z计算为C61H87N5O69S8Na6([M+6Na–10H]4–):596.7651,实测值:596.7657。
5-氨基戊基(2-脱氧-2-磺氨基-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-α-L-艾杜吡喃环草隆基)-(1→4)-(2-脱氧-2-磺氨基-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-α-L-艾杜吡喃环草隆基)-(1→4)-(2-脱氧-2-磺氨基-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-α-L-艾杜吡喃环草隆基)-(1→4)-(2-脱氧-2-磺氨基-α-D-葡萄吡喃糖基)-(1→4)-2-O-磺酸根基-α-L-艾杜吡喃环草隆酸酯(HS8)。1H NMR(600MHz,D2O):δ5.40–5.27(m,7H),5.10(d,J＝2.5Hz,1H),4.93–4.90(m,1H),4.87–4.85(m,1H),4.89–4.87(m,1H),4.48–4.47(m,1H),4.38–4.31(m,4H),4.27–4.20(m,4H),4.19–4.17(m,1H),4.11–4.00(m,5H),3.91–3.80(m,12H),3.78–3.64(m,11H),3.45(t,J＝9.6Hz,1H),3.26–3.20(m,4H),2.99(t,J＝7.9Hz,1H),1.71–1.59(m,4H,CH2连接子),1.50–1.39(m,2H,CH2连接子)；HRMS(ESI):m/z计算为C53H84N5O65S8([M–5H]5–):417.2238,实测值:417.2224。
5-氨基戊基(2-脱氧-2-磺氨基-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-α-L-艾杜吡喃环草隆基)-(1→4)-(2-脱氧-2-磺氨基-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-β-D-葡萄吡喃环草隆基)-(1→4)-(2-脱氧-2-磺氨基-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-α-L-艾杜吡喃环草隆基)-(1→4)-(2-脱氧-2-磺氨基-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-2-O-磺酸根基-β-D-葡萄吡喃环草隆酸酯(HS12)。1H NMR(600MHz,D2O):δ5.68(d,J＝3.6Hz,1H),5.64(d,J＝3.6Hz,1H),5.49–5.46(m,2H),5.30(bs,2H),4.98(d,J＝1.08Hz,1H),4.94(d,J＝1.0Hz,1H),4.66(d,J＝7.7Hz,1H),4.60(d,J＝10.8Hz,1H),4.44–4.38(m,5H),4.38-4.34(m,2H),4.33–4.28(m,2H),4.26(d,J＝10.0Hz,2H),4.24–4.20(m,3H),4.16(dd,J＝8.70,8.19Hz,1H),4.10–4.06(m,2H),4.05–3.98(m,6H),3.98–3.97(m,1H),3.97–3.92(m,4H),3.92(bs,1H),3.91–3.84(m,4H),3.79(t,J＝9.7Hz,1H),3.75–3.70(m,1H),3.61(t,J＝9.7Hz,1H),3.45(dd,J＝10.7,3.7Hz,1H),3.43–3.38(m,3H),3.05(t,J＝7.4Hz,2H),1.76–1.66(m,4H,CH2连接子),1.59–1.49(m,2H,CH2连接子)；13C NMR(150MHz,D2O):δ176.4(C),176.1(C),175.6(C),101.6(CH),101.0(CH),99.9(CH),99.6(CH),97.0(CH),96.7(CH),92.3(CH),92.2(CH),81.1(CH),80.7(CH),78.3(CH),78.0(CH),77.9(CH),77.2(CH),77.0(CH),76.9(CH),76.3(CH),76.1(CH),76.0(CH),74.0(CH),73.8(CH),71.8(CH),71.3(CH),71.1(CH2),70.8(CH),70.7(CH),70.4(CH),69.8(CH),69.5(CH),69.3(CH),68.7(CH),68.3(CH),68.2(CH),67.14(CH2),67.07(CH2),66.5(CH2),64.0(CH),63.6(CH),55.4(CH),55.2(CH),55.0(CH),40.5(CH2),29.0(CH2),27.2(CH2),23.0(CH2)。
5-氨基戊基(2-脱氧-2-磺氨基-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-α-L-艾杜吡喃环草隆基)-(1→4)-(2-脱氧-2-磺氨基-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-α-L-艾杜吡喃环草隆基)-(1→4)-(2-脱氧-2-磺氨基-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-(2-O-磺酸根基-α-L-艾杜吡喃环草隆基)-(1→4)-(2-脱氧-2-磺氨基-6-O-磺酸根基-α-D-葡萄吡喃糖基)-(1→4)-2-O-磺酸根基-α-L-艾杜吡喃环草隆酸酯(HS6)。1H NMR(600MHz,D2O):δ5.43–5.36(m 4H),5.24–5.15(m,4H),5.09–5.04(m,2H),4.76–4.75(m,2H),4.48–4.46(m,1H),4.43–4.29(m,7H),4.28–4.15(m,9H),4.12–3.95(m,8H),3.83–3.71(m,5H),3.69–3.60(m,5H),3.54(t,J＝9.4Hz,1H),3.29–3.19(m,4H),3.01–2.93(m,2H),1.69–1.51(m,4H),1.47–1.39(m,2H)；13C NMR(150MHz,D2O):δ99.0(CH),97.7(CH),97.3(CH),97.2(CH),96.8(CH),76.4(CH),76.3(CH),76.2(CH),76.1(CH),76.0(CH),75.9(CH),75.8(CH),75.4(CH),68.9(CH),68.8(CH),68.6(CH),68.2(CH2),66.4(CH2),66.3(CH2),57.9(CH),39.4(CH2),27.8(CH2),26.2(CH2),22.2(CH2)；HRMS(ESI):m/z计算为C53H73N5O77S12Na4H12([M+4Na–9H]5–):498.7747,实测值:498.7736。
实施例2使用预先涂布实施例一的磺化八糖的磁珠来捕捉胆管癌细胞
按照“材料与方法”章节所述的步骤来建构一种使用实施例一的磺化八糖的磁珠来捕捉癌细胞的微流体***。
在一预测试中，分别将八种细胞株(MMNK-1、SNU-478、HuCCT-1、Huh-28、KKU-100、HepG2、BxPC3与HCT8)注入微***中并与磁珠作用，接着以磁力收集被捕捉的细胞，最后注入血球细胞计数器中计算捕捉率。其结果总结于表一。
表一以实施例一的HS1或HS12来捕捉不同细胞株的捕捉率(％)
在八种细胞株中，HS12与HS1对Huh28细胞(一种胆管癌细胞株)具有高度特异性。HS12与HS1对Huh28细胞的最高捕捉率分别为73±4％与78±14％，其比涂布上皮细胞黏附因子(EpCAM)的磁珠的捕捉率(58±19％)为高。此外，相较于对正常细胞(MMNK-1)和转移性胆管癌细胞(HuCCT-1)的低捕捉率，HS12和HS1甚至更具特异性。因此，HS12和HS1有潜力作为用于检测胆管癌细胞的特异性亲和试剂。
图2说明预先涂布100微体积摩尔浓度的HS1的磁珠可成功地捕捉Huh-28癌细胞。图2(a)为磁珠捕捉之前的Huh-28细胞。在将Huh-28细胞与磁珠充分混合30分钟后，细胞被磁珠包围(图2(b))。在作用30分钟后，通过磁力分离并收集上清液，如图2(c)所示。几乎所有的Huh-28细胞均被磁珠捕捉并分离，且只有极为少数的细胞可在上清液中观察到。这表示大多数Huh-28细胞均被捕获。
虽然实施例于上述文中仅作为示范例之用，然而对于本领域普通技术人员，可以对其进行各种修改。以上说明书、实施例与数据提供对于本发明示范性实施例的结构与用途的完整描述。虽然上述文字已以一定程度的特殊性描述本发明中不同实施例，或参考一或多个单独实施例，但本领域普通技术人员，在不悖离本发明的原理与精神的情形下，当可对其进行各种更动与修饰，因此本发明的保护范围当以附随申请专利范围所界定者为准。
Claims (16)
1.一种用以检测一生物样本中胆管癌细胞(cholangio-cancerous cells)的方法，该方法包含：使该生物样本与一预先涂布式(I)磺化八糖(sulfated octasaccharide)的磁珠接触，
其中，
R1是-NHAc或-NHSO3M；
R2与R3各自为H或-SO3M；以及
M是一单价阳离子，其选自由钠离子、钾离子、锂离子及铵离子所组成的群组；以及
该生物样本与该磁珠之间的结合表示该生物样本中具有胆管癌细胞。
2.如权利要求1所述的方法，其中该式(I)的磺化八糖还与一生物素连接。
3.如权利要求1所述的方法，其中在该式(I)的磺化八糖中，R1是-NHSO3Na；且R2与R3各自为-SO3Na。
4.如权利要求1所述的方法，其中在该式(I)的磺化八糖中，R1是-NHSO3Na，R2与R3各自为H。
5.如权利要求1所述的方法，其中该生物样本是选自由血液、血浆、血清、尿液、痰液、唾液、组织样本、活体组织切片和组织溶解产物所组成的群组。
6.一种微流体芯片，包含：
复数个洗涤缓冲液腔，其分别用以滞留其中的洗涤缓冲液；
复数个捕捉腔，其分别用以将一癌细胞捕捉至一预先涂布癌细胞生物标记的磁珠上；
一废液腔，其用以滞留自该复数个捕捉腔中洗出的未捕捉的癌细胞；以及
复数个微通道，其用以连接该复数个洗涤缓冲液腔、该复数个捕捉腔与该废液腔。
7.如权利要求6所述的微流体芯片，其中该微流体芯片是由一玻璃基板和至少一聚二甲基硅氧烷层所制成。
8.如权利要求7所述的微流体芯片，其中各该捕捉腔是用以分离出一液体样本中与磁珠结合的癌细胞。
9.如权利要求8所述的微流体芯片，其中该磁珠是以式(I)的磺化八糖预先涂布，
其中，
R1是-NHAc或-NHSO3M；
R2与R3各自为H或-SO3M；以及
M是一单价阳离子，其选自由钠离子、钾离子、锂离子及铵离子所组成的群组。
10.如权利要求9所述的微流体芯片，其中该式(I)的磺化八糖还与一生物素连接。
11.如权利要求9所述的微流体芯片，其中在该式(I)的磺化八糖中，R1是-NHSO3Na；且R2与R3各自为–SO3Na。
12.如权利要求9所述的微流体芯片，其中在该式(I)的磺化八糖中，R1是-NHSO3Na，R2与R3各自为H。
13.一种试剂盒，包含：
如权利要求6所述的微流体芯片，
复数个磁珠，其上分别涂布生物标记，
至少一试剂，其用以分析利用如权利要求6所述的微流体芯片所得的生物样本，以及
一使用说明，其用以指示一使用者如何使用该试剂盒；
其中，
该生物标记为式(I)的磺化八糖，
其中，
R1是-NHAc或-NHSO3M；R2与R3各自为H或-SO3M；且M是一单价阳离子，其选自由钠离子、钾离子、锂离子及铵离子所组成的群组。
14.如权利要求13所述的试剂盒，其中在该式(I)的磺化八糖中，R1是-NHSO3Na；且R2与R3各自为-SO3Na。
15.如权利要求13所述的试剂盒，其中在该式(I)的磺化八糖中，R1是-NHSO3Na，R2与R3各自为H。
16.如权利要求13所述的试剂盒，其中该生物样本是血液、血浆、血清、尿液、痰液、唾液、组织、活体组织切片或组织溶解产物。
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| PCT/US2019/019712 WO2019168890A1 (en) | 2018-03-01 | 2019-02-27 | Devices and methods for detecting cancerous cells |
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| WO2007092713A2 (en) * | 2006-02-02 | 2007-08-16 | Trustees Of The University Of Pennsylvania | Microfluidic system and method for analysis of gene expression in cell-containing samples and detection of disease |
| US20110070660A1 (en) * | 2008-03-12 | 2011-03-24 | University Of Virginia Patent Foundation | Detection of polymeric analytes |
| US20110207921A1 (en) * | 2008-09-24 | 2011-08-25 | Shanghai Institute Of Materia Medica, Chinese Academy Of Sciences | Sulfated derivative of gastrodia elata polysaccharide, preparation method and antitumor use thereof |
| US20130157303A1 (en) * | 2010-09-02 | 2013-06-20 | Yasuhiko Tabata | Sulfated polysaccharide capable of binding to growth factor and use thereof |
| WO2015109180A2 (en) * | 2014-01-16 | 2015-07-23 | Academia Sinica | Compositions and methods for treatment and detection of cancers |
| TW202041861A (zh) * | 2019-05-10 | 2020-11-16 | 國立清華大學 | 用於偵測膽管癌細胞的方法 |
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| EP2072133A1 (en) * | 2007-12-20 | 2009-06-24 | Koninklijke Philips Electronics N.V. | Multi-compartment device with magnetic particles |
| CN102625915B (zh) * | 2009-07-14 | 2014-12-31 | 独立行政法人产业技术综合研究所 | 糖蛋白的测定方法、试剂及糖链标记物 |
| JP5686098B2 (ja) * | 2009-09-17 | 2015-03-18 | Jsr株式会社 | アビジンとビオチン誘導体の解離方法及び解離剤 |
| US20130116423A1 (en) * | 2010-04-23 | 2013-05-09 | A. Stewart Campbell | Synthetic Oligosaccharides for Staphylococcus Vaccine |
| US9951149B2 (en) * | 2013-06-17 | 2018-04-24 | The University Of North Carolina At Chapel Hill | Reversible heparin molecules and methods of making and using the same |
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| WO2007092713A2 (en) * | 2006-02-02 | 2007-08-16 | Trustees Of The University Of Pennsylvania | Microfluidic system and method for analysis of gene expression in cell-containing samples and detection of disease |
| US20110070660A1 (en) * | 2008-03-12 | 2011-03-24 | University Of Virginia Patent Foundation | Detection of polymeric analytes |
| US20110207921A1 (en) * | 2008-09-24 | 2011-08-25 | Shanghai Institute Of Materia Medica, Chinese Academy Of Sciences | Sulfated derivative of gastrodia elata polysaccharide, preparation method and antitumor use thereof |
| US20130157303A1 (en) * | 2010-09-02 | 2013-06-20 | Yasuhiko Tabata | Sulfated polysaccharide capable of binding to growth factor and use thereof |
| WO2015109180A2 (en) * | 2014-01-16 | 2015-07-23 | Academia Sinica | Compositions and methods for treatment and detection of cancers |
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