CN107615062B - 具有可调光学性质的水凝胶颗粒以及使用其的方法 - Google Patents
具有可调光学性质的水凝胶颗粒以及使用其的方法 Download PDFInfo
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- CN107615062B CN107615062B CN201680019908.4A CN201680019908A CN107615062B CN 107615062 B CN107615062 B CN 107615062B CN 201680019908 A CN201680019908 A CN 201680019908A CN 107615062 B CN107615062 B CN 107615062B
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Abstract
本申请描述了水凝胶颗粒和它们在细胞计数应用中的用途。本申请描述的水凝胶颗粒是选择性可调的,从而具有至少一种光学性质,其基本上相似于靶细胞的该至少一种光学性质。就此而言,在一个方面中,本申请提供的水凝胶颗粒用作检测样本中靶细胞的标定试剂。
Description
相关申请的交叉引用
本申请要求2015年2月9日提交的美国临时申请62/114,004和2015年6月24日提交的美国临时申请62/184,192的优先权,它们各自的公开内容完全并入本申请以作参考。
背景技术
流式细胞计数法是这样的技术,其可快速分离、计数、和表征个体细胞,其通常用于多种应用的临床和实验室环境。该技术依赖于将光束导向到液体的流体聚焦流上。然后多个检测器瞄准下述点,其中液体流在所述点穿过光束:一个点与光束成一直线(前向散射或FSC),几个点与光束垂直(侧向散射或SSC)。FSC与细胞体积相关,且SSC取决于颗粒的内在复杂性(例如,细胞核的形状,细胞质颗粒的数量和类型或细胞膜表面粗糙度)。由于这些相关性,不同的具体细胞类型表现出不同的FSC和SSC,使得可在流式细胞计数法中区别细胞类型。
但是,确认具体细胞类型的能力依赖于对仪器的适当标定,这是有赖于使用目标细胞类型的纯化细胞的方法。获得这些纯化细胞可能需要昂贵费力的过程,容易带来批次差异。因此,在本领域需要具有可调光学性质的合成组合物,其可以模拟在诸如流式细胞仪中的具体细胞类型。
发明内容
在本发明的一方面,提供了包含聚合的单体并且具有至少一个表面的水凝胶颗粒。水凝胶颗粒具有的至少一种光学性质基本上相似于靶细胞的至少一种光学性质。在一种实施方式中,光学性质是侧向散射波形(SSC),前向散射波形(FSC),荧光发射波形,或其组合。靶细胞可以是用户指定的任何靶细胞。例如,在一种实施方式中,靶细胞是免疫细胞,干细胞或癌细胞。
另一方面,提供了标定用于分析靶细胞的细胞计数装置的方法。在一种实施方式中,所述方法包括向所述装置中插入具有至少一种基本上相似于靶细胞的光学性质的水凝胶颗粒,其中所述水凝胶颗粒包含聚合的单体并且具有至少一个表面。所述方法进一步包括使用所述细胞计数装置测量所述水凝胶颗粒的所述至少一种光学性质。在一种实施方式中,所述至少一种光学性质用作检测样本中的靶细胞的参照物。
再在另一方面,提供了检测样本中靶细胞的方法。所述方法包括向所述装置中插入具有至少一种基本上相似于靶细胞的光学性质的水凝胶颗粒,其中所述水凝胶颗粒包含聚合的单体。所述方法还包括使用所述细胞计数装置测量所述水凝胶颗粒的所述至少一种光学性质。将包含细胞群体的样本插入所述细胞计数装置中,并测量所述群体中的个体细胞的至少一种光学性质。最后,基于所述光学性质测量结果确定所述靶细胞或其群体是否存在于所述样本中。
在本申请提供的方法的一种实施方式中,水凝胶颗粒包含可生物降解的单体。在进一步的实施方式中,可生物降解的单体是单糖,二糖,多糖,肽,蛋白质,或蛋白质结构域。在甚至进一步的实施方式中,可生物降解的单体用丙烯酰胺或丙烯酸酯官能化。
附图说明
图1说明公开的水凝胶颗粒相比于聚苯乙烯小珠的光学性质。
图2描述制备本申请标记的水凝胶颗粒的方法。
图3提供本申请标记的水凝胶颗粒的亮视野图像和荧光图像。
图4说明本申请水凝胶颗粒作为展示多种光学散射性质的细胞类型的标定物的用途。
图5提供显示流式细胞仪的液滴间延迟与水凝胶颗粒直径之间关系的数据(dating)。
图6提供中国仓鼠卵巢细胞(6A和6B)和本申请水凝胶颗粒(6C和6D)的亮视野图像(6A和6C)和荧光图像(6B和6D)。
图7提供数据,其显示人口腔黏膜细胞与包封不同量的DNA的水凝胶颗粒的比较,通过荧光激活细胞分选系统(FACS)测定。
图8提供包封不同浓度的纳米粒子的水凝胶颗粒的数据,其表明侧向散射的调节独立于前向散射。
图9提供用不同百分比聚合物制备的水凝胶颗粒的数据,其表明通过前向散射测量折射率的调节。
图10显示调节成匹配和/或模拟所需细胞种群指标的水凝胶参数的一种实施方式。
图11和12是显示如何调整水凝胶颗粒的前向散射性质、侧向散射性质和表面性质的实施方式的示意图。
图13是各种水凝胶颗粒(A)和(B)和商业血液样本(C)的散点图。
具体实施方式
除非上下文明确指出,否则不定冠词“一个”和“一种”和定冠词“这个”意在包括单数和复数两者。
“至少一种”和“一种或多种”可互换使用,表示该冠词可以包括一个或多于一个所列要素。
除非另有指出,否则应该理解说明书和权利要求书中使用的表达成分、反应条件等的量、比率、和数值性质的所有数目在所有情况下均可由术语“约”修饰。
流式细胞仪的几种临界标定(critical calibration)测量法需要精确的时间分辨率,例如设置激光之间的偏移时间,和计算对象的检测和分选之间的延迟时间。由于仪器内的流体条件,精确设置这些正时参数需要使用尺寸与待分析细胞尺寸一样的标定颗粒。正时标定通常使用具有可变荧光强度的聚苯乙烯小珠进行以标定激发源的响应和设置激光间正时延迟和分选延迟。流式细胞仪也可以使用前向散射和侧向散射信号标定,所述信号是靶样本的尺寸和粒度或复杂性的通用量度。这些标定对于细胞仪的精确性能和对于细胞种群(population)的任何下游分析或分选都是关键的。公开的水凝胶颗粒表现出可调的散射性质,且适宜用作多种哺乳动物或细菌细胞类型的标定试剂。对于临床、食品安全、和研究目的而言,散射是在非均相混合物中区分细胞类型的标准度量。
尽管聚苯乙烯颗粒可用于设置一些应用的激光间延迟和分选延迟,但是很多真核细胞类型都处于商购聚苯乙烯颗粒(1-20μm)的尺寸范围外,使其对于这些靶细胞而言几乎不可能精确标定流式细胞仪。同样,如图1所示,聚苯乙烯颗粒在光学性质上基本上是有限度的,其可具有例如侧向散射,这是细胞复杂性的一般量度。聚苯乙烯颗粒因此在用于流式细胞计数法的两种最重要的被动光学测量上是有限度的:FSC(前向散射),和SSC(侧向散射),它们分别测量靶细胞的尺寸和复杂性。由于聚苯乙烯的这些限度,用户必须依赖于纯化的细胞系来标定实验的荧光强度、激光间延迟,分选延迟、尺寸和细胞复杂性。这是费时费力的过程,会显著增加流式细胞计数法验证和研究管道的成本。最重要的是,这些标定细胞系会带来生物学变动,在解释数据时导致数据存在差异。
此外,当这些仪器用于临床应用例如为分离人调节性T细胞或干细胞而用于下游细胞疗法时,标定流式细胞仪的品质控制(QC)也是关键的考量。FDA规定,在施用给患者之前,必须证实细胞疗法产品的无菌、身份、纯度、和效力(Riley et al.(2009)。Immunity30,pp.656-665)。因此,聚苯乙烯QC颗粒污染细胞种群也是一个问题,因为聚苯乙烯已牵涉到某些癌症中。另外,由QC标准物污染的在施用给患者之后在体内酶降解或消化的细胞种群可能需要克服它们所引起的污染问题。
本发明解决这些和其它需要,如以下讨论。
一方面,提供了包含水凝胶颗粒群体(plurality)的组合物,其中所述水凝胶颗粒群体的个体水凝胶颗粒各自具有的一种或多种光学性质基本上相似于靶细胞的一种或多种光学性质。所述水凝胶颗粒群体的个体水凝胶颗粒各自包含水凝胶,所述水凝胶通过使一种或多种单体聚合、即从而形成均聚物或共聚物来合成。如以下进一步讨论,使用双官能单体允许将水凝胶进一步衍生化,例如,用荧光染料、细胞表面标记物或其抗原表位结合片段、或其组合衍生化。调节成满足/匹配所需细胞子群落指标的水凝胶参数的一个实例在图10中给出。调节水凝胶性质的方法如本申请所述。调整包括水凝胶组分及其浓度等参数范围的能力提供了调节颗粒以模拟广泛细胞例如本申请所述细胞类型之一的能力。
如上提供,一方面,本发明提供个体水凝胶颗粒,其各自具有的一种或多种光学性质基本上相似于靶细胞的一种或多种光学性质。在一种实施方式中,一种或多种光学性质是侧向散射波形,前向散射波形或二级标记物波形,例如荧光标记物波形,例如结合于水凝胶颗粒表面的荧光标记抗体的荧光标记物波形。本申请使用的“基本上相似”表示至少40%相似,至少50%相似,至少60%相似,至少70%相似,至少80%相似,至少90%相似,至少95%相似,至少96%相似,至少97%相似,至少98%相似或至少99%相似。
本发明部分基于以下预料不到的发现,水凝胶颗粒的一种或多种光学性质可以如下独立地调整:改变水凝胶颗粒的组成,例如,改变组合物中初始单体(或共聚单体)的量;改变表面官能化;改变聚合引发剂的量;或改变交联剂的量。例如,可以调整侧向散射(SSC),而不会显著影响前向散射(FSC),反之亦然。此外,可以调节水凝胶颗粒的光学性质(例如折射率),而对颗粒密度不会产生显著应用。这是令人惊奇且有用的特性,因为用作细胞计数方法例如流式细胞计数法或(荧光激活细胞分选系统)FACS中的细胞替代品的水凝胶颗粒需要最小的密度以便于在这些试验中起作用。
另一方面,提供了制备水凝胶颗粒的方法,其中水凝胶颗粒具有的一种或多种光学性质基本上相似于一种或多种靶细胞的光学性质。在一种实施方式中,水凝胶颗粒具有预定的光学性质。在一种实施方式中,光学性质是SSC,FSC,荧光发射,或其组合。
再在另一方面,提供了标定用于分析靶细胞的细胞计数装置的方法。在一种实施方式中,所述方法包括(a)向所述装置中插入具有至少一种基本上相似于靶细胞的光学性质的水凝胶颗粒;b)使用所述细胞计数装置测量所述水凝胶颗粒的所述至少一种光学性质,从而标定用于分析靶细胞的细胞计数装置。细胞计数装置是本领域已知的,并且包括进行流式细胞计数法和FACS的商购装置。
如上提供,在本发明的一方面,提供了包含水凝胶颗粒的群体的组合物。水凝胶是包含大分子三维网络的材料,这种网络使得其当存在水时可溶胀,当不存在水(或减少水的量)时会收缩,但不溶解于水。溶胀,即,吸水,是由于存在连接于大分子网络或分散在大分子网络内的亲水性官能团产生的。相邻大分子间的交联使这些水凝胶在含水介质中不溶解。交联可能是由于化学结合(即,共价键)或物理结合(即,范德华力,氢键结合,离子力等)。合成制备的水凝胶可以如下制备:使单体性物质聚合以形成主链,和使主链与交联剂交联。本申请提及的术语“水凝胶”是指大分子物质,不管其是脱水状态还是水合状态。水凝胶特别有价值的特征是该物质不管是脱水还是水合状态均保持大体形状。因此,如果水凝胶在脱水状态具有近似球形的形状,则其在水合状态也将是球形的。
在一种实施方式中,本申请公开的水凝胶颗粒包含大于约30%的水、大于约40%的水、大于约50%的水、大于约55%的水、大于约60%的水、大于约65%的水、大于约70%的水、大于约75%的水、大于约80%的水、大于约85%的水、大于约90%的水、或大于约95%的水。在另一种实施方式中,水凝胶颗粒的水含量为约10重量%至约95重量%,或约20重量%至约95重量%,或约30重量%至约95重量%,或约40重量%至约95重量%,或约50重量%至约95重量%,或约60重量%至约95重量%,或约70重量%至约95重量%,或约80重量%至约95重量%。
本申请提供的水凝胶为颗粒形式,如下合成:使本申请提供的一种或多种单体聚合。进行合成以形成个体水凝胶颗粒。在一种实施方式中,单体性物质(单体)聚合以形成均聚物。但是,在另一种实施方式中,合成不同单体性单元(即,共聚单体)的共聚物并将其用于本申请提供的方法。在一种实施方式中,用于本申请所述方法和组合物的单体和共聚单体是双官能单体或包括双官能单体(其中使用共聚单体)。在一种实施方式中,水凝胶在交联剂的存在下合成。在进一步的实施方式中,水凝胶在聚合引发剂的存在下合成。
单体的量可以由本发明的用户改变,例如以获得基本上相似于靶细胞的特定光学性质。在一种实施方式中,单体性组分(即,单体,共聚单体,双官能单体,或其组合,例如,各种交联比率的双/丙烯酰胺,烯丙基胺或为二级标记/缀合提供化学官能度的其它共聚单体或藻酸盐)的存在量为水凝胶的10重量%至约95重量%。在进一步的实施方式中,单体性组分的存在量为水凝胶的约15重量%至约90重量%,或水凝胶的约20重量%至约90重量%。
可用于本发明的各种单体和交联化学的实例提供于Thermo Scientific交联技术手册中题为“Easy molecular bonding crosslinking technology”的内容(可查阅于tools.lifetechnologies.com/content/sfs/brochures/1602163-Crosslinking- Reagents-Handbbook.pdf,其公开内容全部并入本申请以作参考,用于所有目的。例如,肼偶联反应(例如,与NHS酯化合物)或EDC偶联反应(例如,与马来酰亚胺化合物)可用于构建本发明的水凝胶。
在一种实施方式中,用于本申请提供的水凝胶的单体是乳酸,乙醇酸,丙烯酸,甲基丙烯酸1-羟基乙基酯,甲基丙烯酸乙基酯,甲基丙烯酸2-羟基乙基酯(HEMA),甲基丙烯酸丙二醇酯,丙烯酰胺,N-乙烯基吡咯烷酮(NVP),甲基丙烯酸甲酯,甲基丙烯酸缩水甘油酯,甲基丙烯酸甘油酯(GMA),甲基丙烯酸甘油酯,乙二醇,富马酸,其衍生化类型,或其组合。
在一种实施方式中,一种或多种以下单体用于本申请以形成本发明的水凝胶:甲基丙烯酸2-羟基乙基酯,甲基丙烯酸羟基乙氧基乙基酯,甲基丙烯酸羟基二乙氧基乙基酯,甲基丙烯酸甲氧基乙基酯,甲基丙烯酸甲氧基乙氧基乙基酯,甲基丙烯酸甲氧基二乙氧基乙基酯,聚(乙二醇)甲基丙烯酸酯,甲氧基-聚(乙二醇)甲基丙烯酸酯,甲基丙烯酸,甲基丙烯酸钠,甲基丙烯酸甘油酯,甲基丙烯酸羟基丙基酯,甲基丙烯酸羟基丙基酯,或其组合。
在另一种实施方式中,一种或多种以下单体用于本申请以形成可调的水凝胶:丙烯酸苯基酯,甲基丙烯酸苯基酯,丙烯酸苄基酯,甲基丙烯酸苄基酯,丙烯酸2-苯基乙基酯,甲基丙烯酸2-苯基乙基酯,丙烯酸2-苯氧基乙基酯,甲基丙烯酸2-苯氧基乙基酯,丙烯酸苯基硫代乙基酯,甲基丙烯酸苯基硫代乙基酯,丙烯酸2,4,6-三溴苯基酯,甲基丙烯酸2,4,6-三溴苯基酯,丙烯酸五溴苯基酯,甲基丙烯酸五溴苯基酯,丙烯酸五氯苯基酯,甲基丙烯酸五氯苯基酯,丙烯酸2,3-二溴丙基酯,甲基丙烯酸2,3-二溴丙基酯,丙烯酸2-萘基酯,甲基丙烯酸2-萘基酯,丙烯酸4-甲氧基苄基酯,甲基丙烯酸4-甲氧基苄基酯,丙烯酸2-苄氧基乙基酯,甲基丙烯酸2-苄氧基乙基酯,丙烯酸4-氯苯氧基乙基酯,甲基丙烯酸4-氯苯氧基乙基酯,丙烯酸2-苯氧基乙氧基乙基酯,甲基丙烯酸2-苯氧基乙氧基乙基酯,N-苯基丙烯酰胺,N-苯基甲基丙烯酰胺,N-苄基丙烯酰胺,N-苄基甲基丙烯酰胺,N,N-二苄基丙烯酰胺,N,N-二苄基甲基丙烯酰胺,N-二苯基甲基丙烯酰胺,N-(4-甲基苯基)甲基丙烯酰胺,N-1-萘基丙烯酰胺,N-4-硝基苯基丙烯酰胺,N-(2-苯基乙基)丙烯酰胺,N-三苯基甲基丙烯酰胺,N-(4-羟基苯基)丙烯酰胺,N,N-甲基苯基丙烯酰胺,N,N-苯基苯基乙基丙烯酰胺,N-二苯基甲基甲基丙烯酰胺,N-(4-甲基苯基)甲基甲基丙烯酰胺,N-1-萘基甲基丙烯酰胺,N-4-硝基苯基甲基丙烯酰胺,N-(2-苯基乙基)甲基丙烯酰胺,N-三苯基甲基甲基丙烯酰胺,N-(4-羟基苯基)甲基丙烯酰胺,N,N-甲基苯基甲基丙烯酰胺,N,N′-苯基苯基乙基甲基丙烯酰胺,N-乙烯基咔唑,4-乙烯基吡啶,2-乙烯基吡啶,如描述于美国专利号6,657,030,其全部并入本申请以作参考,用于所有目的。
合成单体和生物单体都可用于本申请提供的水凝胶,以形成包含合成组分和生物组分(例如,存在于生物分子上的肽、蛋白质、单糖、二糖、多糖、伯胺巯基、羰基、碳水化合物、羧酸)的合成水凝胶、生物水凝胶、或杂合水凝胶。例如,蛋白质、肽或碳水化合物可以用作独立单体以形成水凝胶,该水凝胶包括或不包括合成单体(或聚合物)且与化学相容共聚单体和交联剂组合(参见例如,Thermo Scientific交联技术手册中题为“Easy molecularbonding crosslinking technology”的内容,可查阅于tools.lifetechnologies.com/ content/sfs/brochures/1602163-Crosslinking-Reagents-Handbook.pdf,其公开内容全部并入本申请以作参考,用于所有目的)。相容的交联剂包括但不限于,胺,羧基,和其它反应性化学侧基。可用于本申请所述水凝胶和单体的代表性的反应性基团列在下表1中。
通常,本领域技术人员常规已知的任何形式的聚合化学/方法可以用于形成聚合物。在一些实施方式中,聚合可以通过紫外线诱导的自由基形成和反应级数催化。在其它实施方式中,公开的水凝胶颗粒通过丙烯酰胺的聚合或丙烯酸酯的聚合制备。例如,在一种实施方式中,丙烯酰胺是可聚合碳水化合物衍生化的丙烯酰胺,如描述于美国专利号6,107,365,其公开内容全部并入本申请以作参考,用于所有目的。如本申请所述且如本领域技术人员已知,丙烯酰胺基团与糖的特异性连接便于施用于多种单糖或级数更高的多糖,例如,血清或组织中发现的合成多糖或源自天然来源的多糖例如糖蛋白。
在一种实施方式中,丙烯酸酯官能化的聚(亚乙基)二醇单体用作水凝胶单体。例如,在一种实施方式中,PEG是丙烯酸酯或丙烯酰胺官能化的PEG。
在一些实施方式中,水凝胶颗粒包含与至少一种双官能单体聚合的单官能单体。一个实例包括但不限于,使用丙烯酰胺和双丙烯酰胺(双官能单体)形成聚丙烯酰胺聚合物。在另一种实施方式中,本申请提供的水凝胶颗粒包含与第二双官能单体聚合的双官能单体。一个实例包括但不限于,用包含相容化学物质的混合组合物例如丙烯酰胺、双丙烯酰胺、和包含众多另外化学物质的双丙烯酰胺结构同系物形成聚合物。化学相容单体、双官能单体、和混合组合的反应对于本领域技术而言是显而易见的,且遵循本领域技术人员已知的化学反应原理。(参考Thermo手册和丙烯酰胺聚合手册)。参见,例如,Thermo Scientific交联技术手册中题为“Easy molecular bonding crosslinking technology”的内容(可查阅于tools.lifetechnologies.com/content/sfs/brochures/1602163-Crosslinking- Reagents-Handbook.pdf),和聚丙烯酰胺乳化手册(SNF Floerger,可查阅于snf.com.au/downloads/Emulsion_Handbook_E.pdf),其各自的公开内容完全并入本申请以作参考,用于所有目的。
在一种实施方式中,本申请提供的水凝胶颗粒包含可聚合的单官能单体并且是单官能丙烯酸类单体。用于本申请的单官能丙烯酸类单体的非限制性实例是丙烯酰胺;甲基丙烯酰胺;N-烷基丙烯酰胺,例如N-乙基丙烯酰胺,N-异丙基丙烯酰胺或N-叔丁基丙烯酰胺;N-烷基甲基丙烯酰胺,例如N-乙基甲基丙烯酰胺或N-异丙基甲基丙烯酰胺;N,N-二烷基丙烯酰胺,例如N,N-二甲基丙烯酰胺和N,N-二乙基-丙烯酰胺;N-[(二烷基氨基)烷基]丙烯酰胺,例如N-[3-(二甲基氨基)丙基]丙烯酰胺或N-[3-(二乙基氨基)丙基]丙烯酰胺;N-[(二烷基氨基)烷基]甲基丙烯酰胺,例如N-[3-二甲基氨基)丙基]甲基丙烯酰胺或N-[3-(二乙基氨基)丙基]甲基丙烯酰胺;丙烯酸(二烷基氨基)烷基酯,例如丙烯酸2-(二甲基氨基)乙基酯,丙烯酸2-(二甲基氨基)丙基酯,或丙烯酸2-(二乙基氨基)乙基酯;和甲基丙烯酸(二烷基氨基)烷基酯,例如甲基丙烯酸2-(二甲基氨基)乙基酯。
双官能单体是可与本申请单官能单体聚合以形成本申请所述水凝胶的任何单体,所述双官能单体进一步包含第二官能团,该第二官能团可参与第二反应,例如,荧光团或细胞表面受体(或其结构域)的缀合。
在一些实施方式中,双官能单体选自:烯丙基胺,烯丙基醇,异硫氰酸烯丙基酯,烯丙基氯,和烯丙基马来酰亚胺。
双官能单体可以是双官能丙烯酸类单体。双官能丙烯酸类单体的非限制性实例是N,N′-亚甲基双丙烯酰胺,N,N′-亚甲基双甲基丙烯酰胺,N,N′-亚乙基双丙烯酰胺,N,N′-亚乙基双甲基丙烯酰胺,N,N′-亚丙基双丙烯酰胺和N,N′-(1,2-二羟基亚乙基)双丙烯酰胺。
高阶支化链和直链共聚单体可在聚合物混合物中被取代,以调整折射率同时保持聚合物密度,如描述于美国专利号6,657,030,其全部并入本申请以作参考,用于所有目的。
在一些实施方式中,水凝胶包含调整水凝胶光学性质的分子。以下进一步讨论能够改变水凝胶光学性质的分子。
在一种实施方式中,个体水凝胶颗粒或其群体包含作为水凝胶单体的可生物降解的聚合物。在一种实施方式中,可生物降解的聚合物是基于聚丙交酯(PLA)、聚乙交酯(PGA)、聚己内酯(PCL)的聚(酯),和它们的共聚物。在一种实施方式中,可生物降解的聚合物是碳水化合物或蛋白质,或其组合。例如,在一种实施方式中,单糖、二糖、或多糖(例如,葡萄糖,蔗糖,或麦芽糊精)、肽、蛋白质(或其结构域)用作水凝胶单体。其它可生物降解的聚合物包括PHB-PHV类的聚(羟基玩醇酸酯),另外的聚(酯),和天然聚合物,例如,改性的多(糖)类,例如,淀粉,纤维素,和壳聚糖。在另一种实施方式中,生物相容聚合物是黏附蛋白,纤维素,碳水化合物,淀粉(例如,麦芽糊精,2-羟基乙基淀粉,海藻酸),右旋糖苷,木质素,聚氨基酸,氨基酸,或壳质。这样的可生物降解的聚合物可商购,例如,购自Sigma Aldrich(St.Louis,MO)。
在一种实施方式中,蛋白质仅包含天然氨基酸。但是,本发明并不限于此。例如,自组装人工蛋白和含有非天然氨基酸(例如,结合到非核糖体肽或经合成方法合成引入的那些,参见例如,Zhang et al.(2013)。当前观点在Structural Biology 23,pp.581-587中,其公开内容全部并入本申请以作参考,用于所有目的)、或其蛋白质结构域的蛋白质也可以用作水凝胶单体。可结合到这些组合物中的非天然(非自然)氨基酸的范围是本领域技术人员熟知的(Zhang et al.(2013)。当前观点在Structural Biology 23,pp.581-587中;其完全并入本申请以作参考,用于所有目的)。在一种实施方式中,可生物降解的聚合物用作共聚单体,即,单体混合物中。在一种实施方式中,可生物降解的聚合物是双官能单体。
在一种实施方式中,生物单体用丙烯酰胺或丙烯酸酯官能化。例如,在一种实施方式中,可聚合丙烯酰胺官能化的生物分子是丙烯酰胺或丙烯酸酯官能化的蛋白质(例如,丙烯酰胺官能化的胶原蛋白或官能化的胶原蛋白结构域),丙烯酰胺或丙烯酸酯官能化的肽,或丙烯酰胺或丙烯酸酯官能化的单糖、二糖或多糖。
任何单糖、二糖或多糖(官能化的或相反)可以用作水凝胶单体。在一种实施方式中,丙烯酰胺或丙烯酸酯官能化的单糖、二糖、或多糖用作可聚合的水凝胶单体。在一种实施方式中,结构多糖用作可聚合的水凝胶单体。在进一步的实施方式中,结构多糖是阿拉伯木聚糖,纤维素,壳质或胶质。在另一种实施方式中,海藻酸(海藻酸盐)用作可聚合的水凝胶单体。再在另一种实施方式中,糖胺多糖(GAG)在本申请提供的水凝胶中用作可聚合的单体。在进一步的实施方式中,GAG是硫酸软骨素,硫酸皮肤素,硫酸角质素,肝素,硫酸肝素或透明质酸(在本领域也称为透明质酸酶或透明质酸盐/酯),其用作可聚合水凝胶单体。相容生物单体和它们的反应性化学的另外的范围是本领域技术人员已知的并且遵循一般化学反应性原理。
可以结合的生物相容单体的另外的范围是本领域已知的,参见,例如不可降解的生物相容单体公开于Shastri(2003)。Current Pharmaceutical Biotechnology 4,pp.331-337,其完全并入本申请以作参考,用于所有目的。其它单体提供于de MoraesPorto(2012).Polymer Biocompatibility,Polymerization,Dr.Ailton De Souza Gomes(Ed.),ISBN:978-953-51-0745-3;InTech,DOI:10.5772/47786;Heller et al.(2010).Journal of Polymer Science Part A:Polymer Chemistry 49,pp.650-661;FinalReport for Biocompatible Materials(2004),The Board of the BiocompatibleMaterials and the Molecular Engineering in Polymer Science programmes,ISBN91-631-4985-0,其各自的公开内容完全并入本申请以作参考。
在一种实施方式中,用于本申请所述水凝胶的生物相容单体包括而甲基丙烯酸乙二醇酯(EGDMA),甲基丙烯酸2-羟基乙基酯(HEMA),甲基丙烯酸甲酯(MMA),甲基丙烯酰氧基甲基三甲基硅烷(TMS-MA),N-乙烯基-2-吡咯烷酮(N-VP),苯乙烯,或其组合。
用于本发明的天然存在的水凝胶包括可得自天然来源的各种多糖,例如植物,藻类,真菌,酵母,海洋无脊椎动物和节肢动物。非限制性实例包括琼脂糖,右旋糖苷,壳质,基于纤维素的化合物,淀粉,衍生化淀粉等。这些将通常具有重复的葡萄糖单元作为多糖主链的主要部分。这些多糖的交联化学是本领域已知的,参见例如Thermo Scientific交联技术手册中题为“Easy molecular bonding crosslinking technology”的内容,(可查阅于tools.lifetechnologies.com/content/sfs/brochures/1602163-Crosslinking- Reagents-Handbook.pdf)。
在一种实施方式中,透明质酸用作水凝胶单体(或作为单一单体或作为共聚单体)。在一种实施方式中,透明质酸例如用丙烯酸酯或丙烯酰胺官能化。透明质酸是由通过交替的β-1,4糖苷键和β-1,3糖苷键连接在一起的N-乙酰葡糖胺和葡糖醛酸的二糖重复单元构成的高分子量GAG。在人体中,透明质酸盐/酯存在于几种软结缔组织中,包括皮肤,脐带,滑膜液,和玻璃体。因此,在一种实施方式中,当需要模拟皮肤细胞、脐带细胞或玻璃体细胞时,在一种实施方式中,透明质酸用作水凝胶单体。制造水凝胶颗粒的方法描述于Xuet al.(2012).Soft Matter.8,pp.3280-3294,其公开内容完全并入本申请以作参考,用于所有目的。如本申请所述,根据所需的交联剂和用于形成水凝胶颗粒的其它单体,透明质酸可以用各种反应性臂(reactive handles)衍生化。
再在其它实施方式中,壳聚糖、由无规分布的β-(1-4)-连接的D-葡糖胺(脱乙酰基单元)和N-乙酰基-D-葡糖胺(乙酰化单元)组成的直链多糖用作水凝胶单体(或作为单独单体或作为共聚单体)。
用作水凝胶单体或共聚单体的其它多糖包括但不限于,琼脂,琼脂糖,海藻酸,微藻多糖酸(alguronic acid),α-葡聚糖,支链淀粉,直链淀粉,阿拉伯木聚糖,β-葡聚糖,胼胝质,卡苏兰(capsulan),角叉菜胶多糖(例如,κ、ι或λ类),纤维糊精,动物纤维素,纤维素,壳质,壳聚糖,金藻昆布多糖,凝胶多糖,环糊精,α-环糊精,糊精,聚蔗糖,果聚糖,岩藻依聚糖,半乳葡甘露聚糖,半乳甘露聚糖,半乳糖胺半乳糖,结冷胶,葡聚糖,葡甘露聚糖,葡萄醛酸木聚糖,糖萼,糖原,半纤维素,同多糖,羟丙甲纤维素,艾考糊精,菊粉,开菲尔多糖,海带多糖,蘑菇多糖,果聚糖多糖,地衣多糖,甘露聚糖,混联葡聚糖,裸藻淀粉,果胶酸,果胶,喷他淀粉,植物糖原,平菇葡聚糖,聚葡萄糖,多糖肽,金属卟啉,普鲁兰多糖,裂褶菌多醣,左旋糖,西佐糖,文莱胶,黄原胶,木聚糖,木葡聚糖,酵母聚糖,或其组合。如全文所述,根据所需交联剂和/或用于水凝胶的另外的共聚单体,多糖可以是进一步官能化的。例如,在一种实施方式中,一种或多种本申请所述的多糖用丙烯酸酯或丙烯酰胺官能化。
在一种实施方式中,个体水凝胶颗粒或其群体包含肽、蛋白质、蛋白质结构域、或其组合作为水凝胶单体或其群体。在进一步的实施方式中,蛋白质是结构蛋白,或其结构域,例如,例如蚕丝蛋白,弹性蛋白,肌联蛋白或胶原蛋白,或其结构域。在一种实施方式中,蛋白质是细胞外基质(ECM)成分(例如,胶原蛋白,弹性蛋白,蛋白多糖)。在甚至进一步的实施方式中,结构蛋白是胶原蛋白。再在进一步的实施方式中,胶原蛋白是胶原蛋白I型,胶原蛋白II型,或胶原蛋白III型或其组合。在另一种实施方式中,水凝胶单体包括蛋白多糖。在进一步的实施方式中,蛋白多糖是核心蛋白多糖,双糖链蛋白多糖,睾丸蛋白聚糖,双库尼茨抑制剂,纤维调节素,光蛋白聚糖,或其结构域。
在另一种实施方式中,丙烯酸酯官能化的结构蛋白水凝胶单体用作本申请提供的水凝胶的组分(例如,丙烯酸酯官能化的蛋白质或蛋白质结构域,例如,蚕丝蛋白,弹性蛋白,肌联蛋白,胶原蛋白,蛋白多糖,或其官能化的结构域)。在进一步的实施方式中,丙烯酸酯官能化的结构蛋白水凝胶单体包括蛋白多糖,例如,核心蛋白多糖,双糖链蛋白多糖,睾丸蛋白聚糖,双库尼茨抑制剂,纤维调节素,光蛋白聚糖,或其结构域。
在一种实施方式中,PEG单体和寡肽可以模拟细胞外基质蛋白质用于本申请提供的水凝胶,例如,用乙烯基砜官能化的多臂PEG、整合素结合肽和双-半胱氨酸基质金属蛋白酶肽进行,如由Lutolf et al.(2003).Proc.Natl.Acad.Sci.U.S.A.100,5413-5418所述,其完全并入本申请以作参考,用于所有目的。在该特定的实施方式中,水凝胶通过二硫醇化寡肽和PEG上的乙烯基砜基团之间的Michael类型加成反应形成。可以结合到本申请的另外的相容性化学物质的范围是本领域技术人员显而易见的并且遵循一般化学反应性原理,参见例如Thermo Scientific交联技术手册中题为“Easy molecular bonding crosslinkingtechnology”的内容(可查阅于tools.lifetechnologies.com/content/sfs/brochures/ 1602163-Crosslinking-Reagents-Handbook.pdf)。
天然蛋白质中的其它生物活性结构域也可以用作水凝胶单体或其部分。例如,细胞粘附整合素结合结构域、控释亲和力结合结构域或谷氨酰胺转胺酶交联结构域可以用于本申请提供的水凝胶。制备这些水凝胶的详情可见Martino et al.(2009).Biomaterials30,1089;Martino et al.(2011).Sci.Trans.Med.3,100ra89;Hu and Messersmith(2003).J.Am.Chem.Soc.125,14298,其各自完全并入本申请以作参考,用于所有目的。
在一种实施方式中,重组DNA方法用于产生蛋白质,该蛋白质设计为响应pH温度的变化而胶凝,例如,通过Petka et al.(1998).Science 281,pp.389-392所述的方法进行,其完全并入本申请以作参考,用于所有目的。简言之,蛋白质由位于水溶性聚电解质段侧面的末端亮氨酸拉链结构域组成。在近乎中性的水性溶液中,末端结构域的卷曲螺旋聚集体形成三维水凝胶聚合物网络。
可用于使本申请提供的水凝胶交联的常用交联剂包括但不限于二甲基丙烯酸乙二醇酯(EGDMA),二甲基丙烯酸四甘醇酯,和N,N′-15-亚甲基双丙烯酰胺。可以使用的另外的交联剂的范围是本领域技术人员显而易见的并且遵循一般化学反应性原理,参见例如Thermo Scientific交联技术手册中题为“Easy molecular bonding crosslinkingtechnology”的内容(可查阅于tools.lifetechnologies.com/content/sfs/brochures/ 1602163-Crosslinking-Reagents-Handbook.pdf)。
在一种实施方式中,水凝胶的聚合由过硫酸盐或催化自由基形成的等效引发剂引发。相容引发剂的范围是本领域技术人员已知的并且遵循一般化学反应性原理,参见例如Thermo Scientific交联技术手册中题为“Easy molecular bonding crosslinkingtechnology”的内容(可查阅于tools.lifetechnologies.com/content/sfs/brochures/ 1602163-Crosslinking-Reagents-Handbook.pdf)。过硫酸盐可以是任何水溶性过硫酸盐。水溶性过硫酸盐的非限制性实例是过硫酸铵和碱金属过硫酸盐。碱金属包括锂,钠和钾。在一些实施方式中,过硫酸盐是过硫酸铵或过硫酸钾。在进一步的实施方式中,本申请提供的水凝胶的聚合由过硫酸铵引发。
水凝胶的聚合可以由可催化聚合不稳定的化学侧基的形成的促进剂加速。可用促进剂的范围是本领域技术人员已知的并且遵循一般化学反应性原理,参见例如ThermoScientific交联技术手册中题为“Easy molecular bonding crosslinking technology”的内容(可查阅于tools.lifetechnologies.com/content/sfs/brochures/1602163- Crosslinking-Reagents-Handbook.pdf)。在一种实施方式中,促进剂是叔胺。叔胺可以是任何水溶性叔胺。在一种实施方式中,促进剂用于聚合反应且为N,N,N′,N′-四甲基乙二胺,3-二甲基氨基)丙腈,或N,N,N′,N′-四甲基乙二胺(TEMED)。在另一种实施方式中,促进剂用于聚合反应且为偶氮二(异丁腈)(AIBN)。
如上讨论,用于本申请组合物和方法的水凝胶可以包括本申请所述的任何单体性单元和交联剂,且在一方面,作为水凝胶颗粒通过使液滴制备(参见,例如,图2)。制备多个液滴(包括流体液滴和僵化液滴)微流体方法是本领域技术人员已知的,且描述于美国专利公开号2011/0218123和美国专利号7,294,503,各自全部并入本申请以作参考,用于所有目的。这样的方法提供包含第一流体且基本上由第二流体包围的多个液滴,其中第一流体和第二流体基本上不溶混(例如,包含基本上由油基液体包围的水基液体液滴)。
多个流体液滴(例如,使用微流体装置制备)可以是多分散的(例如,具有一系列不同尺寸),或在一些情况下,流体液滴可以是单分散的或基本上单分散的,例如,具有直径的均匀分布,例如,使得不多于约10%、约5%、约3%、约1%、约0.03%、或约0.01%的液滴具有的平均直径大于平均直径的约10%、约5%、约3%、约1%、约0.03%、或约0.01%。如本申请使用的液滴的群落的平均直径是指液滴的直径的算术平均值。可以例如通过光散射技术测量颗粒的平均直径。在一种实施方式中,如下调整水凝胶颗粒的平均直径:例如改变微流体装置的通道内第一和第二流体的液流的流速,或改变微流体装置的通道的体积。
另外,本申请提供水凝胶颗粒的群落,其包含多个水凝胶颗粒,其中水凝胶颗粒的群落基本上是单分散的。
术语微流体是指包括至少一个流体通道的装置、设备或系统,所述流体通道的横截面尺寸小于1mm,且长度与垂直于通道的最大横截面尺寸之比为至少约3∶1。包括微流体通道的微流体装置特别适于制备多个单分散液滴。
可用于本发明的微流体系统的非限制性实例公开于美国专利申请公开号2006/0163385;美国专利申请公开号2005/0172476;美国专利申请公开号2007/000342;国际专利申请公开号WO2006/096571;美国专利申请公开号2007/0054119;美国专利号7,776,927;和国际专利申请公开号WO2006/078841,各自全部并入本申请以作参考,用于所有目的。
液滴尺寸与微流体通常尺寸有关。微流体通道可以具有任何尺寸,例如,垂直于液流的最大尺寸为小于约5mm或2mm,或小于约1mm,或小于约500μm,小于约200μm,小于约100μm,小于约60μm,小于约50μm,小于约40μm,小于约30μm,小于约25μm,小于约10μm,小于约3μm,小于约1μm,小于约300nm,小于约100nm,小于约30nm,或小于约10nm。
液滴尺寸可以通过调整相对流速来调节。在一些实施方式中,液滴直径等于通道宽度,或在通道宽度的约10%、15%、20%、30%、40%、50%、60%、70%、80%、90%、或100%内。
公开的水凝胶颗粒的尺寸基本上相似于由其形成的小滴。因此,在一些实施方式中,水凝胶的直径为小于约1μm,小于约2μm,小于约5μm,小于约10μm,小于约15μm,小于约20μm,小于约25μm,小于约30μm,小于约35μm,小于约40μm,小于约45μm,小于约50μm,小于约60μm,小于约70μm,小于约80μm,小于约90μm,小于约100μm,小于约120μm,小于约150μm,小于约200μm,小于约250μm,小于约300μm,小于约350μm,小于约400μm,小于约450μm,小于约500μm,小于约600μm,小于约800μm,或小于1000μm。在一些实施方式中,水凝胶的直径为大于约1μm,大于约2μm,大于约5μm,大于约10μm,大于约15μm,大于约20μm,大于约25μm,大于约30μm,大于约35μm,大于约40μm,大于约45μm,大于约50μm,大于约60μm,大于约70μm,大于约80μm,大于约90μm,大于约100μm,大于约120μm,大于约150μm,大于约200μm,大于约250μm,大于约300μm,大于约350μm,大于约400μm,大于约450μm,大于约500μm,大于约600μm,大于约800μm,或大于1000μm。在一种实施方式中,水凝胶颗粒的直径为5μm至100μm。
在一些实施方式中,公开的水凝胶颗粒的形状为球形。
在一些实施方式中,公开的水凝胶颗粒不包含琼脂糖。
在一种实施方式中,水凝胶颗粒通过悬浮液聚合进行,该聚合法在本领域也称为成珠聚合、珠状聚合、或成粒聚合(参见Elbert(2011)。Acta Biomater.7,pp.31-56,其完全并入本申请以作参考,用于所有目的)。在悬浮液聚合中,单体不溶于连续相,例如单体水性溶液在油连续相中。在悬浮液聚合中,聚合引发在富含单体的液滴内进行,且在任何时候需要针对每个液滴存在大于一个自由基才能进行。在一种实施方式中,单体相包括可以是双官能单体或多种单体物质(共聚单体,其可以是多种双官能单体)的单体。在一种实施方式中,单体相包括引发剂和/或交联剂。
乳液聚合也可以用于形成本申请所述的水凝胶颗粒。但是,类似于悬浮液聚合,在乳液聚合中,单体在连续相中具有差的溶解性,聚合引发在单体液体之外进行(参见Elbert(2011).Acta Biomater.7,pp.31-56,其完全并入本申请以作参考,用于所有目的)。在乳液聚合实施方式中,引发剂引起溶解于连续相中的单体(或共聚单体)或在存在表面活性剂时包含在胶束中的单体的链增长。
在另一种实施方式中,水凝胶颗粒通过沉淀聚合形成,例如描述于Elbert(2011).Acta Biomater.7,pp.31-56,其完全并入本申请以作参考,用于所有目的。沉淀聚合是一种利用单体和聚合物溶解度之差来产生微粒子的技术。具体地,已知较大的聚合物链的溶解度通常小于较小的聚合物链。因此,针对具有的分子量,相分离可能是有利的。沉淀聚合最初作为在单一相、均匀系统中的溶液聚合开始。在一种实施方式中,在聚合开始以后,立即存在相对较高浓度的聚合物链,利于通过成核作用发生相分离。随着聚合的进行,聚合物链的浓度变低,在可出现新颗粒的成核之前,现存颗粒捕获链。因此,颗粒的成核仅在反应开始后即刻出现片刻,这在一种实施方式中使颗粒的尺寸分布较窄。另外的方法包括但不限于光刻粒子形成(Helgeson et al.(2011).Curr.Opin.Colloid.Interface Sci.16,pp.106-117,其完全并入本申请以作参考,用于所有目的),膜乳化(例如,通过Nanomi B.V.(Netherlands)所述的微筛乳化(micosieve emulsification)工艺技术进行),和微通道乳化(Sugiura et al.(2002).Languimir 18,pp.5708-5712,其完全并入本申请以作参考),和本体乳化(SNF Floerger,可查阅于snf.com.au/downloads/Emulsion_Handbook_E.pdf,其完全并入本申请以作参考)。
在一种实施方式中,水凝胶颗粒在具有两个在单体水性溶液的中心流处交汇的油通道的微流体装置内形成。在该实施方式中,液滴在两个通道和中心流的界面处形成,打破油包水乳液中的液滴。在一种实施方式中,一旦形成液滴,它们在聚合之前稳定化,例如,通过向油相中添加表面活性剂稳定化。但是,在另一种实施方式中,液滴在聚合之前不稳定化。在一种实施方式中,单体的聚合如下触发:在形成初始液滴之后,向油通道之一或两者添加促进剂(例如,N,N,N′,N′-四甲基乙二胺)。
如上提供的单体水性溶液可以包括单一单体物质或多种单体物质。单体水性溶液可以包括共聚单体、双官能单体或其组合。在一种实施方式中,单体或单体的群体可以包括双官能单体,例如,上述单体之一。如下所述,共聚单体可以用于调整前向散射或侧向散射,例如,通过调节水凝胶颗粒的折射率进行。
在一种实施方式中,单体水性溶液的中心流包含交联剂,例如,N,N′-双丙烯酰胺。在进一步的实施方式中,除了单体之外,单体水性溶液的中心流还包含交联剂和促进剂。再在进一步的实施方式中,单体水性溶液包含引发剂,例如氧化剂,如过硫酸铵。
前向散射经由调节凝胶的折射率来调整,折射率的调节通过添加共聚单体丙烯酸烯丙基酯和甲基丙烯酸烯丙基酯实现(也参见图11和12)。前向散射也可以用包含足够光学分辨率/尺寸/密度的侧向散射纳米粒子调节,包括但不限于,二氧化硅和/或PMMA颗粒的较高密度胶态悬浮体。液滴的侧向散射如下调节:在聚合之前,将二氧化硅纳米粒子和/或PMMA(聚(甲基丙烯酸甲酯))粒子的胶态悬浮体(~100nm)添加到中心水相(图11和12)。
在一种实施方式中,小珠、多个小珠、生物分子、或多个生物分子嵌埋(包封)在水凝胶颗粒内。在一种实施方式中,包封的小珠或生物分子用于模拟靶细胞的一个或多个细胞内细胞器,或当细胞吞没颗粒后模拟细胞本身。在一种实施方式中,包封或嵌埋小珠或生物分子在水凝胶颗粒形成时完成。例如,小珠可以按适当的浓度悬浮,从而使平均一个小珠嵌埋/包封在单个水凝胶颗粒中。小珠悬浮液可以包含例如在单体的水性溶液内。类似地,生物分子或生物分子的混合物可以结合到单体的水性溶液中以包封一个或多个生物分子。
或者,一旦例如通过上述方法形成水凝胶颗粒,在一种实施方式中,其可以进一步如下处理:例如,将小珠、多个小珠、生物分子或多个生物分子嵌埋在水凝胶颗粒内。
因此,在本发明的一方面,提供了包含嵌埋物质的水凝胶。
在一种实施方式中,嵌埋物质是嵌埋分子,例如生物分子。生物分子可以是单一物质或多种不同物质。例如,蛋白质、肽、碳水化合物、核酸或其组合可以包封在本发明水凝胶颗粒内。此外,不同的核酸分子(例如,具有不同序列或核酸类型,例如基因组DNA,信使RNA或DNA-RNA杂合体)可以由本发明水凝胶颗粒包封。这些可以包含任何蛋白质或核酸作为包含不稳定化学侧基(或可以由供应商改性(例如,Integrated DNA Technology化学侧基改性)的生物材料的两种形式。这样的侧基与共聚单体组合物中常见的反应化学物质(例如丙烯酸酯化学物质,NHS酯,伯胺,铜催化的点击化学(Sharpless))相容。可用的包含相容化学物质的嵌埋分子的范围是本领域技术人员可理解的。
在一种实施方式中,制造水凝胶颗粒的不同子群落,每种具有不同浓度的生物分子。在进一步的实施方式中,生物分子是核酸,蛋白质,细胞内离子例如酸钙(或用户选择的其它生物分子,例如,钙)。在另一种实施方式中,制造水凝胶颗粒的不同子群落,每种具有不同浓度的药物物质。在一种实施方式中,药物物质是生物分子(即,生物药物,抗体药物,抗体药物缀合物,蛋白质/酶,肽,非核糖体肽,或有关分子)或小分子合成药物(例如,I/II/III型聚酮化合物,具有生物活性性质的非核糖体肽,或通常由本领域技术人员归类的其它小分子实体)。
在这点上,本发明特别适用于确定试验分辨率,其中将细胞中的各核酸或蛋白质内容物染色。在一种实施方式中,本申请提供的水凝胶颗粒的不同群落用已知的不同量的细胞内物质例如核酸或蛋白质包封。将个体水凝胶颗粒中的细胞内物质染色,通过细胞计数装置测量多种群落的个体水凝胶的荧光。这使得能够产生标准曲线以确立细胞内试验的灵敏度和动态量程。一旦确立,可以通过细胞仪运行样本以检测是否存在靶细胞,和定量各靶细胞中细胞内物质的量。在一种实施方式中,嵌埋的物质是传染病生物标记物,例如传染病生物标记物数据库(IDBD,参见Yang et al.(2008)IDBD:Infectious DiseaseBiomarker Database.Nucleic Acid Res.36,pp.D455-D460,其完全并入本申请以作参考,用于所有目的)中的传染病生物标记物之一。在进一步的实施方式中,传染病生物标记物是以下的生物标记物:胃肠道感染,呼吸道感染,神经系统感染,泌尿生殖器感染,病毒感染,出血热,动物传染病,虫媒病毒,抗生素耐药性或生物恐怖。在进一步的实施方式中,病毒感染是埃博拉病毒感染。
在一种实施方式中,本申请提供的方法用于确定细胞核酸定量试验的灵敏度和/或动态量程。在该实施方式中,对于样本,询查样本内(是否存在)的细胞类型,以及细胞内的细胞核酸的量。
在另一种实施方式中,本发明提供确定细胞内蛋白质定量试验的分辨率和/或灵敏度的手段。在一种实施方式中,水凝胶颗粒以各种浓度包封已知量的蛋白质,然后用适当的蛋白质抗体染色。测量各种颗粒的荧光以确定试验的灵敏度和/或动态量程。然后将该荧光值与从样本中的细胞获得的荧光值相比较,以确定是否存在靶细胞,以及其是否包含细胞内蛋白质,以及蛋白质的量。
在一种实施方式中,调节个体水凝胶颗粒,使其具有的至少一种光学性质基本上相似于存在于母血中存在的循环肿瘤细胞或胎儿细胞。个体颗粒用已知量的待分析生物分子嵌埋。对于特定细胞类型,颗粒用于产生生物分子检测试验的标准曲线。
如上提供,在本发明的一方面,提供包含嵌埋的物质的水凝胶。在一种实施方式中,嵌埋的物质是一个小珠或多个小珠。在一种实施方式中,水凝胶颗粒用单个小珠嵌埋。在另一种实施方式中,对于个体水凝胶,嵌埋的小珠在多个水凝胶颗粒中的平均数为1。
在将一个或多个小珠嵌埋进水凝胶颗粒的情况中,在一种实施方式中,为了流式细胞计数试验的品质控制,将一个或多个小珠的光学性质水凝胶颗粒的FSC和SSC性质组合使用。例如,在一种实施方式中,嵌埋的小珠用作标定流式细胞仪系统的控制物,包括激光源,光学性质,和流量。在另一种实施方式中,嵌埋的小珠用作定量样本例如特定细胞中的荧光量的手段。关于这点,各种浓度的嵌埋的小珠可以用于产生荧光标准曲线,以确定细胞是否表达某些标记物以及处于何种表达水平。
在一种实施方式中,直径为约1μm至约3μm、约2μm至约4μm或约3μm至约7μm的小珠嵌埋在本申请提供的水凝胶中。例如,在一种实施方式中,小珠的直径为约3μm至约3.5μm。在进一步的实施方式中,小珠是荧光小珠。在另一种实施方式中,小珠的直径为约1μm至约2.5μm或约1.5μm至约3μm。在进一步的实施方式中,小珠是荧光小珠且可以是内部染色的或在其表面染色。在甚至进一步的实施方式中,荧光小珠是内部染色的。不希望受任何理论限制,认为内部染色使荧光团与可引起荧光输出变化的环境相互作用隔绝。
如上提供,在一种实施方式中,嵌埋的小珠是荧光小珠,在进一步的实施方式中,荧光小珠是内部染色的。本领域技术人员能够选择适当的荧光团来与嵌埋的小珠联用。在一种实施方式中,小珠用以下荧光染料中的一种或多种衍生化:6-羧基-4′,5′-二氯-2′,7′-二甲氧基荧光素琥珀酰亚胺酯;5-(和-6)-羧基曙红;5-羧基荧光素;6羧基荧光素;5-(和-6)-羧基荧光素;S-羧基荧光素-二-(5-羧基甲氧基-2-硝基苄基)醚,-丙氨酸-甲酰胺、或琥珀酰亚胺酯;5-羧基荧光素琥珀酰亚胺酯;6-羧基荧光素琥珀酰亚胺酯;5-(和-6)-羧基荧光素琥珀酰亚胺酯;5-(4,6-二氯三嗪基)氨基荧光素;2′,7′-二氟荧光素;曙红-5-异硫氰酸酯;赤藓红5-异硫氰酸酯;6-(荧光素-5-甲酰氨基)己酸或琥珀酰亚胺酯;6-(荧光素-5-(和-6)-甲酰氨基)己酸或琥珀酰亚胺酯;荧光素-S-EX琥珀酰亚胺酯;荧光素-5-异硫氰酸酯;荧光素-6-异硫氰酸酯;488羧酸、或琥珀酰亚胺酯;488异硫氰酸酯;488-X琥珀酰亚胺酯;500羧酸;500羧酸、琥珀酰亚胺酯或三乙铵盐;514羧酸;514羧酸或琥珀酰亚胺酯;罗丹明绿TM羧酸、琥珀酰亚胺酯或盐酸盐;罗丹明绿TM羧酸、三氟乙酰胺或琥珀酰亚胺酯;罗丹明绿TM-X琥珀酰亚胺酯或盐酸盐;罗德尔绿TM羧酸,N,O-二-(三氟乙酰基)或琥珀酰亚胺酯;二-(4-羧基哌啶基)砜罗丹明或二(琥珀酰亚胺酯);5-(和-6)羧基萘并荧光素,5-(和-6)羧基萘并荧光素琥珀酰亚胺酯;5-羧基罗丹明6G盐酸盐;6-羧基罗丹明6G盐酸盐,5-羧基罗丹明6G琥珀酰亚胺酯;6-羧基罗丹明6G琥珀酰亚胺酯;5-(和-6)-羧基罗丹明6G琥珀酰亚胺酯;5-羧基-2′,4′,5′,7′-四溴砜荧光素琥珀酰亚胺酯或二-(二异丙基乙基铵)盐;5-羧基四甲基罗丹明;6-羧基四甲基罗丹明;5-(和-6)-羧基四甲基罗丹明;5-羧基四甲基罗丹明琥珀酰亚胺酯;6-羧基四甲基罗丹明琥珀酰亚胺酯;5-(和-6)-羧基四甲基罗丹明琥珀酰亚胺酯;6-羧基-X-罗丹明;5-羧基-X-罗丹明琥珀酰亚胺酯;6-羧基-X-罗丹明琥珀酰亚胺酯;5-(和-6)-羧基-X-罗丹明琥珀酰亚胺酯;5-羧基-X-罗丹明三乙铵盐;丽丝胺TM罗丹明B磺酰氯;孔雀绿;异硫氰酸酯;单(磺基琥珀酰亚胺酯);21羧酸或琥珀酰亚胺酯;7羧酸或琥珀酰亚胺酯;罗丹明红TM-X琥珀酰亚胺酯;6-(四甲基罗丹明-5-(和-6)-甲酰氨基)己酸;琥珀酰亚胺酯;四甲基罗丹明-5-异硫氰酸酯;四甲基罗丹明-6-异硫氰酸酯;四甲基罗丹明-5-(和-6)-异硫氰酸酯;磺酰基;磺酰氯;-X STP酯或钠盐;-X琥珀酰亚胺酯;-X琥珀酰亚胺酯;和X-罗丹明-5-(和-6)异硫氰酸酯,商购自Invitrogen的染料,其包括但不限于FL;TMR STP酯;TR-X STP酯;630/650-X STP酯;650/665-X STP酯;6-二溴-4,4-二氟-5,7-二甲基-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酸琥珀酰亚胺酯;4,4-二氟-4-硼杂-3a,4a-二氮杂-对称-引达省-3,5-二丙酸;4,4-二氟-5,7-二甲基-4-硼杂-3a,4a-二氮杂-对称-引达省-3-戊酸;4,4-二氟-5,7-二甲基-4-硼杂3a,4a-二氮杂-对称-引达省-3-戊酸琥珀酰亚胺酯;4,4-二氟-5,7-二甲基-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酸;4,4-二氟-5,7-二甲基-4-硼杂-3a,4a二氮杂-对称-引达省-3-丙酸琥珀酰亚胺酯;4,4二氟-5,7-二甲基-4-硼杂-3a,4a-二氮杂-对称-引达省-3丙酸;磺基琥珀酰亚胺酯或钠盐;6-((4,4-二氟-5,7-二甲基-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酰基)氨基)己酸;6-((4,4-二氟-5,7-二甲基-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酰基)氨基)己酸或琥珀酰亚胺酯;N-(4,4-二氟-5,7-二甲基-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酰基)磺基丙氨酸,琥珀酰亚胺酯或三乙铵盐;6-4,4-二氟-1,3-二甲基-5-(4-甲氧基苯基)-4-硼杂3a,4a4,4-二氟-5,7-二苯基-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酸;4,4-二氟-5,7-二苯基-4-硼杂3a,4a-二氮杂-对称-引达省-3-丙酸琥珀酰亚胺酯;4,4-二氟-5-苯基-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酸;琥珀酰亚胺酯;6-((4,4-二氟-5-苯基-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酰基)氨基己酸或琥珀酰亚胺酯;4,4-二氟-5-(4-苯基-1,3-丁二烯基)-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酸琥珀酰亚胺酯;4,4-二氟-5-(2-吡咯基)-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酸琥珀酰亚胺酯;6-(((4,4-二氟-5-(2-吡咯基)-4-硼杂-3a,4a-二氮杂-对称-引达省-3-基)苯乙烯氧基)乙酰基)氨基己酸或琥珀酰亚胺酯;4,4-二氟-5-苯乙烯基-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酸;4,4-二氟-5-苯乙烯基-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酸;琥珀酰亚胺酯;4,4-二氟-1,3,5,7-四甲基-4-硼杂-3a,4a二氮杂-对称-引达省-8-丙酸;4,4-二氟-1,3,5,7-四甲基-4硼杂-3a,4a-二氮杂-对称-引达省-8-丙酸琥珀酰亚胺酯;4,4-二氟-5-(2-噻吩基)-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酸琥珀酰亚胺酯;6-(((4-(4,4-二氟-5-(2-噻吩基)-4-硼杂-3a,4a二氮杂对称-引达省-3-基)苯氧基)乙酰基)氨基)己酸或琥珀酰亚胺酯;和6-(((4,4-二氟-5-(2-噻吩基)-4-硼杂-3a,4a-二氮杂-对称-引达省-3-yl)苯乙烯氧基)乙酰基)氨基己酸或琥珀酰亚胺酯,商购自Invitrogen的Alexa fluor染料,其包括但不限于Alexa350羧酸;Alexa430羧酸;Alexa488羧酸;Alexa532羧酸;Alexa546羧酸;Alexa555羧酸;Alexa568羧酸;Alexa594羧酸;Alexa633羧酸;Alexa647羧酸;Alexa660羧酸;和Alexa680羧酸,商购自Amersham-Pharmacia Biotech的花青染料,其包括但不限于Cy3 NHS酯;Cy 5 NHS酯;Cy5.5 NHS酯;和Cy7 NHS酯。
可用于本发明的其它荧光团在以下表2中给出。
可商购的小珠包括但不限于由Bangs Laboratories,Inc,Sperhotech Inc.,Thermo Scientific,Inc.以及同等的供应商出售的那些,其可与本申请所述的水凝胶颗粒组合使用。本领域技术人员根据试验可以选择适当小珠直径、荧光发射和/或激发光谱和/或荧光强度的小珠。例如,与蓝色、红色、或紫外激光联用的品质控制小珠可以嵌埋进本申请提供的一个或多个水凝胶颗粒中。例如,对于蓝色激光(目录号A-16500(2.5μm),A-16503(6.0μm))、红色激光(目录号A-16501(2.5μm),A-16504(6.0μm))或紫外激光(目录号A-16502(2.5μm),A-16505(6.0μm)),AlignflowTM流式细胞仪定位小珠可以嵌埋在本申请提供的一个或多个水凝胶颗粒中。
在一种实施方式中,可在在365nm-650nm范围内任何波长激发的荧光小珠嵌埋在水凝胶颗粒中。在一种实施方式中,小珠是“彩虹粒子”,其包含荧光团的混合物,例如4个荧光团,5个荧光团,6个荧光团,7个荧光团或8个荧光团。鉴于此,用户根据待问询的荧光团选择用哪一波长来激发粒子。彩虹粒子可商购,例如,商购自BD Biosciences(目录号556298(中波程FL1荧光),556286(6色,3.0-3.4μm),556288(6色,6.0-6.4μm),559123(8色))和各种直径的Spherotech(例如,目录号RCP20-5(4色),RCP-30-5(6个峰),RCP-30-5A(8个峰)
细胞分选设置小珠可以嵌埋在本申请提供的一个或多个水凝胶颗粒中。在一种实施方式中,细胞分选设置小珠在尺寸、发射波长、和强度上近似于生物样本,并且可以用于标定流式细胞仪的细胞分选系统,包括激光源、光学性质、和流量。在一种实施方式中,细胞分选设置小珠嵌埋在一个或多个水凝胶颗粒中并且可用于紫外激光、蓝色激光、绿色/黄色激光或红色激光。在一种实施方式中,当使用绿色激光时,嵌埋的小珠在570nm激发并在575nm发射,但是也可以在488nm激发。商购细胞分选设置小珠购自,例如LifeTechnologies(目录号C-16506(紫外激光),C-16508(蓝色激光),C-16509(绿色-黄色激光),C-16507(红色激光))。
补偿控制小珠也可以嵌埋在本申请提供的一个或多个水凝胶颗粒中。精确补偿对于流式细胞计数法中的有效多色分析是重要参数。但是,基于细胞的补偿控制并不是完全有效的,因为很多抗原并不是高表达的,且依稀染色的细胞可能导致补偿设置不准确。
在一种实施方式中,补偿控制小珠包括荧光抗体缀合物捕获能力(正补偿小珠)或者是惰性的(负补偿小珠)。将补偿小珠与缀合荧光团的人、小鼠、大鼠、仓鼠、或兔子抗体混合;两种组分提供了明显的高信号正控制与适度的负群落,该负群落然后可用于适当地设置补偿,而不管细胞在实际实验中的浓度如何。一旦将抗体与小珠混合,则抗体嵌埋在本申请提供的一个或多个水凝胶颗粒中。商购补偿小珠购自,例如Life Technologies(目录号A-10344,A-10389,A10497,A10513)和Spherotech(目录号CMIg-P-08-2K,CMIg-P-30-2K,CMIg-P-50-3K,CMIg-P-70-3K)。
在一种实施方式中,含有嵌埋/包封小珠的水凝胶颗粒用作细胞试验的参照物,例如,吞噬试验,细胞毒性试验,运动性试验,细胞活性试验等。吞噬作用是下述过程,通过该过程细胞吞没固体颗粒,形成称为吞噬体的内囊。鉴于此,可以调节水凝胶颗粒,使其具有一种或多种基本上相似于在吞噬细胞吞没颗粒之前和之后吞噬细胞的光学性质。因此,在一种实施方式中,本申请提供的水凝胶颗粒用作吞噬试验的控制颗粒。在进一步的实施方式中,(i)水凝胶颗粒的一种或多种光学性质基本上相似于在吸收颗粒之前的吞噬细胞,和(ii)第二水凝胶颗粒的一种或多种光学性质基本上相似于在吸收颗粒之后的吞噬细胞。鉴于此,对于测量由吞噬细胞吸收的颗粒,产生控制。
在一种实施方式中,吞噬细胞是专业吞噬细胞。在另一种实施方式中,吞噬细胞是非专业吞噬细胞(即,消耗濒死细胞和外来有机体的细胞)。在进一步的实施方式中,非专业吞噬细胞是上皮细胞,内皮细胞,成纤维细胞或间充质细胞。在一种实施方式中,调解水凝胶颗粒,使其具有的一种或多种光学性质基本上相似于以下表3中列出的专业吞噬细胞(在颗粒吸收之前和/或之后)。
在一种实施方式中,由诸如核酸或小珠等物质嵌埋的本发明的水凝胶颗粒群体用作基因组细胞计数试验的控制试剂。鉴于此,特定染色体、RNA序列和/或DNA序列的特定数目的拷贝可以由嵌埋的物质模拟。水凝胶颗粒然后可以用作有待探测基因信息的样板的控制物,例如染色体的拷贝数目,RNA序列的拷贝数目和/或DNA序列的拷贝数目。
流式细胞计数法的反卷积的三种主要模式是颗粒的两种被动光学性质(前向散射,FSC,对应于折射率,或RI;和侧向散射,SSC)和存在于给定细胞类型表面上的生物标记物。因此,允许本申请水凝胶颗粒对于这三种模式模拟具体细胞类型的组合物用于为流式细胞计数法提供合成的强效的标定剂。
在一种实施方式中,公开的水凝胶颗粒的折射率(RI)大于约1.10,大于约1.15,大于约1.20,大于约1.25,大于约1.30,大于约1.35,大于约1.40,大于约1.45,大于约1.50,大于约1.55,大于约1.60,大于约1.65,大于约1.70,大于约1.75,大于约1.80,大于约1.85,大于约1.90,大于约1.95,大于约2.00,大于约2.10,大于约2.20,大于约2.30,大于约2.40,大于约2.50,大于约2.60,大于约2.70,大于约2.80,或大于约2.90。
在另一种实施方式中,公开的水凝胶颗粒的折射率(RI)为约1.10至约3.0,或约1.15至约3.0,或约1.20至约3.0,或约1.25至约3.0,或约1.30至约3.0,或约1.35至约3.0,或约1.4至约3.0,或约1.45至约3.0,或约1.50至约3.0,或约1.6至约3.0,或约1.7至约3.0,或约1.8至约3.0,或约1.9至约3.0,或约2.0至约3.0。
在一些实施方式中,公开的水凝胶颗粒的折射率(RI)小于约1.10,小于约1.15,小于约1.20,小于约1.25,小于约1.30,小于约1.35,小于约1.40,小于约1.45,小于约1.50,小于约1.55,小于约1.60,小于约1.65,小于约1.70,小于约1.75,小于约1.80,小于约1.85,小于约1.90,小于约1.95,小于约2.00,小于约2.10,小于约2.20,小于约2.30,小于约2.40,小于约2.50,小于约2.60,小于约2.70,小于约2.80,或小于约2.90。
最有意义的是将公开的水凝胶颗粒的SSC相比于靶细胞进行测量。在一些实施方式中,公开的水凝胶颗粒的SSC在靶细胞SSC的30%内、在其25%内、在其20%内、在其15%内、在其10%内、在其5%内、或在其1%内,通过细胞计数装置测得。
在一种实施方式中,如下调整水凝胶颗粒的SSC:将高折射率分子(或其群体)结合到水凝胶中。在一种实施方式中,将高折射率分子配置在水凝胶颗粒中,且在进一步的实施方式中,高折射率分子是硅胶,丙烯酸烷基酯,甲基丙烯酸烷基酯或其组合。因此,在一些实施方式中,公开的水凝胶颗粒丙烯酸烷基酯和/或甲基丙烯酸烷基酯。在一种实施方式中,调节单体的浓度,以进一步调节水凝胶颗粒的折射率。
丙烯酸烷基酯或甲基丙烯酸烷基酯可以在烷基中包含1至18个碳原子、1至8个碳原子、或2至8个碳原子,例如甲基,乙基,正丙基,异丙基,正丁基,异丁基或叔丁基,2-乙基己基,庚基或辛基。烷基可以是支化或直链的。
高折射率分子也可以包括乙烯基芳烃例如苯乙烯和甲基苯乙烯,其在芳环上任选取代有烷基例如甲基、乙基或叔丁基,或取代有卤素,例如氯苯乙烯。
在一些实施方式中,FSC如下调整:调节单体在组合物中存在的百分比,从而改变水凝胶形成过程中存在的水含量。在一种实施方式中,当使用单体和共聚单体时,调解单体和共聚单体的比率以改变水凝胶颗粒的前向散射性质。这在图11和图12中都有显示。
最有意义的是将公开的水凝胶颗粒的FSC相比于靶细胞进行测量。在一些实施方式中,公开的水凝胶颗粒的FSC在靶细胞FSC的30%内、在其25%内、在其20%内、在其15%内、在其10%内、在其5%内、或在其1%内,通过细胞计数装置测得。
FSC涉及颗粒体积,因此可以通过改变颗粒直径调整,如本申请所述。通常,已经观察到,相比于小的物体,大的物体折射折射更多的光,从而得到高的前向散射信号(反之亦然)。因此,在一种实施方式中,改变颗粒直径以调整水凝胶颗粒的FSC性质。例如,在一种实施方式中,水凝胶颗粒直径的增大是通过在流体形成过程中利用较大的微流体通道改变的。
SSC可如下设计:将纳米粒子包封在水凝胶内,以模拟靶细胞中的细胞器。在一些实施方式中,公开的水凝胶颗粒包含一种或多种类型的纳米粒子,其选自:聚甲基丙烯酸甲酯(PMMA)纳米粒子,聚苯乙烯(PS)纳米粒子,和二氧化硅纳米粒子。也参见图11和12,其表明添加各种浓度的纳米粒子使得可调整颗粒的侧向散射。不希望受理论限制,选择性调节本申请所述水凝胶的前向散射和侧向散射两者的能力可提供强效平台来模拟众多细胞类型。
尽管本发明主要描述光学性质的改性,但是本发明并不限于此。例如,可以制造和调整水凝胶颗粒,从而调节颗粒的容量,例如,从而标定库尔特计数器。在一种实施方式中,水凝胶颗粒的容量通过改变水凝胶单体在组合物中的量来调节。例如,聚苯胺,聚乙炔;聚亚苯基亚乙烯基;聚吡咯(X=NH)和聚噻吩(X=S)共聚单体;和聚苯胺(X=NH/N)和聚亚苯基硫醚(X=S)共聚单体浓度均可调节以改变容量。在一种实施方式中,提高这些单体的一种或多种的浓度,以提高水凝胶颗粒的容量。
在一些实施方式中,与具有相同直径的聚苯乙烯小珠相比,公开的水凝胶颗粒具有的材料模量性质(例如,弹性)更加极为相似于靶细胞的所述性质。
在水凝胶颗粒形成之后,可以将一个或多个颗粒表面官能化,例如,从而模拟靶细胞或标记靶细胞的一种或多种光学性质。官能化的水凝胶颗粒也可以包括嵌埋的小珠或物质例如生物分子,如上所述。在一种实施方式中,一个或多个水凝胶颗粒用一种或多种荧光染料、一种或多种细胞表面标记物(或其抗原表位结合区)、或其组合官能化。在一种实施方式中,水凝胶颗粒通过使至少一种双官能单体聚合形成,且在形成之后,水凝胶颗粒包括一个或多个可用于进一步连接细胞表面标记物、细胞表面标记物的抗原表位结合区、荧光染料、或其组合的官能团。在一种实施方式中,自由官能团是胺基,羧基,羟基或其组合。根据所需的官能化作用,应该理解的是,可以使用多种双官能单体,例如,以使用不同的化学方法和不同的分子将颗粒官能化。
水凝胶颗粒可以使用本领域已知的任何荧光染料官能化,包括TheHandbook-A Guide to Fluorescent Probes and LabelingTechnologies中列出的荧光染料,其全部并入本申请以作参考,用于所有目的。官能化可以通过包含自由胺基的化合物例如烯丙基胺介导,所述化合物可被引入所用的双官能单体中以形成水凝胶,如上讨论。
可用于将本申请所述水凝胶颗粒的表面官能化的已知荧光染料的非限制性实例包括:6-羧基-4′,5′-二氯-2′,7′-二甲氧基荧光素琥珀酰亚胺酯;5-(和-6)-羧基曙红;5-羧基荧光素;6羧基荧光素;5-(和-6)-羧基荧光素;S-羧基荧光素-二-(5-羧基甲氧基-2-硝基苄基)醚,-丙氨酸-甲酰胺、或琥珀酰亚胺酯;5-羧基荧光素琥珀酰亚胺酯;6-羧基荧光素琥珀酰亚胺酯;5-(和-6)-羧基荧光素琥珀酰亚胺酯;5-(4,6-二氯三嗪基)氨基荧光素;2′,7′-二氟荧光素;曙红-5-异硫氰酸酯;赤藓红5-异硫氰酸酯;6-(荧光素-5-甲酰氨基)己酸或琥珀酰亚胺酯;6-(荧光素-5-(和-6)-甲酰氨基)己酸或琥珀酰亚胺酯;荧光素-S-EX琥珀酰亚胺酯;荧光素-5-异硫氰酸酯;荧光素-6-异硫氰酸酯;488羧酸、或琥珀酰亚胺酯;488异硫氰酸酯;488-X琥珀酰亚胺酯;500羧酸;500羧酸、琥珀酰亚胺酯或三乙铵盐;514羧酸;514羧酸或琥珀酰亚胺酯;罗丹明绿TM羧酸,琥珀酰亚胺酯或盐酸盐;罗丹明绿TM羧酸、三氟乙酰胺或琥珀酰亚胺酯;罗丹明绿TM-X琥珀酰亚胺酯或盐酸盐;罗德尔绿TM羧酸,N,O-二-(三氟乙酰基)或琥珀酰亚胺酯;二-(4-羧基哌啶基)砜罗丹明或二(琥珀酰亚胺酯);5-(和-6)羧基萘并荧光素,5-(和-6)羧基萘并荧光素琥珀酰亚胺酯;5-羧基罗丹明6G盐酸盐;6-羧基罗丹明6G盐酸盐,5-羧基罗丹明6G琥珀酰亚胺酯;6-羧基罗丹明6G琥珀酰亚胺酯;5-(和-6)-羧基罗丹明6G琥珀酰亚胺酯;5-羧基-2′,4′,5′,7′-四溴砜荧光素琥珀酰亚胺酯或二-(二异丙基乙基铵)盐;5-羧基四甲基罗丹明;6-羧基四甲基罗丹明;5-(和-6)-羧基四甲基罗丹明;5-羧基四甲基罗丹明琥珀酰亚胺酯;6-羧基四甲基罗丹明琥珀酰亚胺酯;5-(和-6)-羧基四甲基罗丹明琥珀酰亚胺酯;6-羧基-X-罗丹明;5-羧基-X-罗丹明琥珀酰亚胺酯;6-羧基-X-罗丹明琥珀酰亚胺酯;5-(和-6)-羧基-X-罗丹明琥珀酰亚胺酯;5-羧基-X-罗丹明三乙铵盐;丽丝胺TM罗丹明B磺酰氯;孔雀绿;异硫氰酸酯;单(磺基琥珀酰亚胺酯);21羧酸或琥珀酰亚胺酯;7羧酸或琥珀酰亚胺酯;罗丹明红TM-X琥珀酰亚胺酯;6-(四甲基罗丹明-5-(和-6)-甲酰氨基)己酸;琥珀酰亚胺酯;四甲基罗丹明-5-异硫氰酸酯;四甲基罗丹明-6-异硫氰酸酯;四甲基罗丹明-5-(和-6)-异硫氰酸酯;磺酰基;磺酰氯;-X STP酯或钠盐;-X琥珀酰亚胺酯;-X琥珀酰亚胺酯;和X-罗丹明-5-(和-6)异硫氰酸酯。
用于本申请所述的水凝胶颗粒的荧光染料的其它实例包括但不限于,商购自Invitrogen的染料,包括但不限于FL;TMR STP酯;TR-X STP酯;630/650-X STP酯;650/665-X STP酯;6-二溴-4,4-二氟-5,7-二甲基-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酸琥珀酰亚胺酯;4,4-二氟-4-硼杂-3a,4a-二氮杂-对称-引达省-3,5-二丙酸;4,4-二氟-5,7-二甲基-4-硼杂-3a,4a-二氮杂-对称-引达省-3-戊酸;4,4-二氟-5,7-二甲基-4-硼杂3a,4a-二氮杂-对称-引达省-3-戊酸琥珀酰亚胺酯;4,4-二氟-5,7-二甲基-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酸;4,4-二氟-5,7-二甲基-4-硼杂-3a,4a二氮杂-对称-引达省-3-丙酸琥珀酰亚胺酯;4,4-二氟-5,7-二甲基-4-硼杂-3a,4a-二氮杂-对称-引达省-3丙酸;磺基琥珀酰亚胺酯或钠盐;6-((4,4-二氟-5,7-二甲基-4-硼杂-3a,4a-二氮杂-对称-引达省-3丙酰基)氨基)己酸;6-((4,4-二氟-5,7-二甲基-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酰基)氨基)己酸或琥珀酰亚胺酯;N-(4,4-二氟-5,7-二甲基-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酰基)磺基丙氨酸,琥珀酰亚胺酯或三乙铵盐;6-4,4-二氟-1,3-二甲基-5-(4-甲氧基苯基)-4-硼杂3a,4a4,4-二氟-5,7-二苯基-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酸;4,4-二氟-5,7-二苯基-4-硼杂3a,4a-二氮杂-对称-引达省-3-丙酸琥珀酰亚胺酯;4,4-二氟-5-苯基-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酸;琥珀酰亚胺酯;6-((4,4-二氟-5-苯基-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酰基)氨基)己酸或琥珀酰亚胺酯;4,4-二氟-5-(4-苯基-1,3-丁二烯基)-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酸琥珀酰亚胺酯;4,4-二氟-5-(2-吡咯基)-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酸琥珀酰亚胺酯;6-(((4,4-二氟-5-(2-吡咯基)-4-硼杂-3a,4a-二氮杂-对称-引达省-3-基)苯乙烯氧基)乙酰基)氨基己酸或琥珀酰亚胺酯;4,4-二氟-5-苯乙烯基-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酸;4,4-二氟-5-苯乙烯基-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酸;琥珀酰亚胺酯;4,4-二氟-1,3,5,7-四甲基-4-硼杂-3a,4a二氮杂-对称-引达省-8-丙酸;4,4-二氟-1,3,5,7-四甲基-4硼杂-3a,4a-二氮杂-对称-引达省-8-丙酸琥珀酰亚胺酯;4,4-二氟-5-(2-噻吩基)-4-硼杂-3a,4a-二氮杂-对称-引达省-3-丙酸琥珀酰亚胺酯;6-(((4-(4,4-二氟-5-(2-噻吩基)-4-硼杂-3a,4a二氮杂对称-引达省-3-基)苯氧基)乙酰基)氨基)己酸或琥珀酰亚胺酯;和6-(((4,4-二氟-5-(2-噻吩基)-4-硼杂-3a,4a-二氮杂-对称-引达省-3-基)苯乙烯氧基)乙酰基)氨基己酸或琥珀酰亚胺酯。
在一种实施方式中,用于衍生化一个或多个水凝胶颗粒的表面的荧光染料包括但不限于,商购自Invitrogen的Alexa fluor染料,其包括但不限于Alexa350羧酸;Alexa430羧酸;Alexa488羧酸;Alexa532羧酸;Alexa546羧酸;Alexa555羧酸;Alexa568羧酸;Alexa594羧酸;Alexa633羧酸;Alexa647羧酸;Alexa660羧酸;和Alexa680羧酸。在另一种实施方式中,用于本申请所述水凝胶颗粒和方法的荧光染料包括商购自Amersham-Pharmacia Biotech的花青染料,其包括但不限于Cy3 NHS酯;Cy5 NHS酯;Cy5.5NHS酯;和Cy7 NHS酯。
本领域技术人员可根据水凝胶颗粒的所需光谱激发和发射性质选择适宜的一种或多种染料。
水凝胶颗粒,在一种实施方式中用一种或多种细胞表面标记物(参见,例如,表4和7-8)或其片段(例如,在跨膜蛋白的情况中为其细胞外部分)衍生化,例如如下进行:将一种或多种细胞表面标记物,其细胞外部分或配套结合区经自由胺、存在于水凝胶颗粒表面上的自由胺、自由羧基和/或自由羟基连接于颗粒。用染料或细胞表面分子将水凝胶颗粒官能化也可以通过连接物例如链霉亲和素/生物素缀合物进行。
根据靶细胞,个体水凝胶颗粒科员用一种或多种细胞表面标记物或其片段(例如,在跨膜蛋白的情况下为其细胞外部分)衍生化,以进一步模拟靶细胞的结构性质。以下给出的表4和7-8列出根据靶细胞可用于将水凝胶颗粒衍生化的细胞表面标记物的非限制性清单。尽管提供的是细胞表面标记物,但是应该理解的是,细胞表面标记物的部分,例如受体结合部分、配体结合部分、或标记物的细胞外部分也可以用于将水凝胶颗粒衍生化(在自由官能团,如上所述)。也参见图11和12,其表明用例如细胞表面受体将水凝胶表面改性连同选择性调节FSC和/或SSC使得能够制造具有所需特性的水凝胶颗粒。
细胞类型包括但不限于各种细胞系,例如CHO,HEK-293,BHK-21,NS0,MDCK,VERO,MRC-S,W1-38和Sp2/0小鼠骨髓瘤(杂交瘤)。表5和表6各自提供了用于本申请所述的水凝胶颗粒的其它细胞类型。
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
15-脂肪氧合酶1
160 kD精神丝中肤
200 kD精神丝重肤
2H2
3G11唾液神经节苷酯抗原
4E-BP1
磷酸化4E-BP1(Thr37/46)
5-甲基胞苷
5HT3A受体
5T4
68kDa神经丝轻肤
7.1
70kD神经丝轻肤
A20
A2B5
AAK1
ABCA1
ABCA7
ABCB4
ABCB5
ABCC10
ABCC11
ABCG1
ABI2
ABIN3
ABL2
Abraxas
ACAA1
ACADM
ACAT2
ACBD3
ACD
ACE2
乙酰辅酶A羧化酶
乙酰辅酶A合酶
乙酰化的赖氨酸
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
顺乌头酸酶2
ACOT12
ACSA2
ACSF2
ACSM5
Act1
激活分子8(B细胞)
IB型激活素A受体
IIB型激活素A受体
ACTN3
ACY1
ACY3
ADA
ADAM12
ADE2
腺苷A1受体
腺苷A2aR
腺病毒
腺病毒纤维单体和三聚物
腺病毒六邻体蛋白
腺苷酸激酶1
腺苷酸琥珀酸裂解酶
ADFP
ADH1B
ADH6
ADH7
ADI1
脂联素
脂联素受体2
脂肪甘油三酯脂酶
ADP核糖基化因子
ADP-核糖基转移酶2.2基因
铁氧还蛋白
AF10
AFAP1
AFP
AG2
AGAP1
AGPAT5
AGR2
AHSG
AICDA
AID
AIF
AIM-2
Aiolos
AIPL1
AIRE
AK3
AK3L1
AK5
Akt
Akt(pS473)
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
Akt(pT308)
Akt1
Akt2
Akt3
自蛋白
醇脱氢酶
醛还原酶
ALDH1A1
ALDH1L1
ALDH2
ALDH3A1
ALDH3A2
ALDH5A1
ALDH6A1
ALDH7A1
ALDOB
缩醛酶B
Alexa Fluor 405/级联蓝
Alexa Fluor 488
ALG2
Alix
变应素1
α1抗胰蛋白酶
α1连环蛋白
α1钠钾ATP酶
α2连环蛋白
α2聚球蛋白
α肌动蛋白1
α肌动蛋白2
α辅肌动蛋白
α辅肌动蛋白2
α辅肌动蛋白3
α辅肌动蛋白4
α衔接蛋白
α内收
αB晶状体蛋白
α胞衬蛋白
α互联蛋白
α突触核蛋白
ALS1
AMACR
氨肤酶P
AML1
Amphiphysin
ANAPC2
AND1
雄激素受体
血管紧张素I
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
血管紧张素II受体2
血管紧张素III
ANKRD53
膜联蛋白IV
膜联蛋白V
ANP
Anti-Kudoa thrysites
布氏抗-T.原周期蛋白酶(procyclin)(GPEET)
布氏抗-T.原周期蛋白酶(磷酸化的GPEET)
抗球蛋白(Coombs)
抗凝血酶III
AP2M1
AP2S1
APAF1
APBB3
APC
APC-1
APC-10
APC-11
APC-2
APC-3
APC-5
APC-7
APC-8
APE1
APG12
APG3
APG5
APG7
APMAP
Apo-2.7
Apo-2.7(7A6)
ApoE
ApoE4
APOER2
载脂蛋白AI
载脂蛋白AII
载脂蛋白AIV
载脂蛋白B
载脂蛋白CIII
载脂蛋白D
载脂蛋白E
载脂蛋白F
载脂蛋白H
载脂蛋白J
载脂蛋白L1
载脂蛋白M
凋亡的嗜中性粒细胞
APP
水通道蛋白1
水通道蛋白5
ARF1
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
ARF5
ARFGAP1
ARFRP1
Argonaute-1
ARH
ARHGAP25
ARHGAP4
ARL11
ARL5B
ARPC5
Artemis
芳基烃受体
ASB-1
ASCC1
ASCC2
ASGPR
去唾液酸-GM1
ASK1
天冬酰胺合酶
Ataxin 1
ATF1
ATF2
ATG4A
ATG9A
ATIC
太平洋鲑鱼Ig
ATM
ATP柠檬酸裂解酶
ATP1B3
ATP5A
ATP5H
ATP5J
ATP5O
ATP6V0D1
ATP6V1B1
ATPB
ATRIP
Aurora A
磷酸化Aurora A(Thr288)
Aurora B
磷酸化Aurora B(Thr232)
AVEN
禽流感A神经氨酸酶
亲和素
虫漆脂2
Axl
B细胞和激活的T细胞
B细胞
B细胞亚基
B细胞(泛反应性)
B淋巴细胞抗体[UCH-B1]
b-內腓肤
磷酸化B-Raf(Thr598/Ser601)
B18R
B7-H4
BACE1
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
BACE2
BACH1
杆状病毒包膜gp64蛋白
BAG1
BAG2
BAG3
BAG4
BAIAP2
BAK
BAMBI
BAP31
BAP37
基底细胞细胞角蛋白
嗜碱性粒细胞
Bassoon
BATF
Bax
BCAR1
BCAR2
BCKD复合物E2亚基
Bcl-10
Bcl-2
Bcl-2(pS70)
Bcl-2样12
Bcl-2样2
Bcl-22
Bcl-2A1
Bcl-3
Bcl-6
Bcl-xL
Bcl-XS/L
BCR
BCSC1
BDH2
BDKRB2
BDNF
Beclin1
斑萎蛋白3
β2肾上腺素受体
B3肾上腺素受体
β3钠钾ATP酶
β肌动蛋白
β视紫红质抑制蛋白1
β视紫红质抑制蛋白2
β连环蛋白
β连环蛋白(npaa 27-37)
β连环蛋白(npaa 35-50)
β连环蛋白(pS45)
B营养不良蛋白聚糖
β半乳糖苷酶
B半乳糖苷酶融合蛋白
B突触核蛋白
β2微球蛋白
BHMT
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
Bid
双糖链蛋白多糖
胆红素氧化酶
Bim
BimL
BIN1
BIN3
蛋白生物素
BiP
BLBP
Blimp-1
BLK
BLNK
BLNK(pY84)
血型A抗原
血型AB抗原
血型B抗原
血型H ab抗原
血型H ab抗原/n抗原
血型H抑制剂
血型Lewis a
血型M抗原
血型N抗原
水华综合征
BM1
BMAL1
BMI1
Bmk
BMP15
BMP4
BMP7
BMPR1A
BMPR2
BMX
bMyc
BNIP2
BNIP3
BNIP3L
BOB1
BORA
Borealin
包柔氏螺旋体
BPI
BRaf
BRCA1
BRCC36
BRD3
BrdU
BRF1
BRG1
BRN3A
Btk
Btk(pY551)/Itk(pY511)
BTLN-2
BTN1A1
Bu1
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
Bu1a
Bu1a/Bu1b
Bu1b
BubR1
Bu1b
丁酰胆碱酯酶
C肤
C反应蛋白
C1抑制剂
C15orf40
C16orf72
C1orf50
C1Q
C1QA
C1QB
C1QC
C1QG
C1r
C1s
C20orf30
C20orf43
C21orf56
C21orf59
C2orf43
C3
C3aR
C3b
C3c
C3d
C4
C4结合蛋白
C4b
C4c
C4d
C4orf42
C5
C5aR1
C5L2
C6
C6orf64
C8A/B/G
C9
C9orf41
CA125
CA19.9
CAB39
CACNA1S
CACNA2
CACNG1
CAD
钙粘蛋白1
钙粘蛋白10
钙粘蛋白11
钙粘蛋白7
钙粘蛋白8
钙粘蛋白9
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
钙粘蛋白E
钙粘蛋白H
钙粘蛋白K
钙粘蛋白P
钙粘蛋白R
CAKC末端
CAKN末端
磷酸化CAK(Ser164/Thr170)
钙结合蛋白
钙调磷酸酶A
降血钙素受体
钙敏感受体
钙调结合蛋白
钙粒蛋白A
钙粒蛋白B
钙调蛋白
钙连接蛋白-ER膜标记物
钙蛋白酶1
钙蛋白酶2
钙蛋白酶9
钙蛋白酶S1(小亚基)
钙蛋白酶抑制蛋白
钙调蛋白
钙网蛋白
钙网膜蛋白
隐钙素2
CaMKI
CaMKII
磷酸化CaMKII(Thr286)
CamKIV
CAMLG
cAMP蛋白激酶催化亚基
大麻素受体I
大麻素受体II
CAP-G2
CAP18
CAP2
CAP3
碳酸酐酶I
碳酸酐酶IX
羧酯酶1
羧肤酶A1
羧肤酶A2
CARD11
CARD8
CARD9
心肌肌钙蛋白T
CARKL
CARM1
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
半胱天冬酶1
半胱天冬酶10
半胱天冬酶11
半胱天冬酶12
半胱天冬酶2
半胱天冬酶2L
半胱天冬酶3
半胱天冬酶4
半胱天冬酶5
半胱天冬酶6
半胱天冬酶7
半胱天冬酶8
半胱天冬酶9
过氧化氢酶
儿茶酚-O-甲基转移酶
组织蛋白酶D
组织蛋白酶K
组织蛋白酶L
小窝蛋白1
小窝蛋白1(pY14)
小窝蛋白2
Cbl
CBP
CBWD1
CBX1
cCbl(pY700)
cCbl(pY774)
CCDC98
CCK4
CCL11
CCL17
CCL18
CCL19-Fc
CCL20
CCL21
CCL25
CCL3
CCL5
CCL6
CCNB1IP1
CCR10
CCR11
CCRD6
CCRL2
CD1
CD1.1
CD10
CD100
CD101
CD102
CD103
CD104
CD105
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
CD106
CD107a
CD107b
CD108
CD109
CD11
CD110
CD111
CD112
CD113
CD114
CD115
CD116
CD117
CD118
CD119
CD11a
CD11a,应变多态性
CD11a/CD18
CD11b
CD11b/c
CD11c
CD11d
CD120a
CD120b
CD121a
CD121b
CD122
CD123
CD124
CD125
CD126
CD127
CD129
CD13
CD130
CD131
CD132
CD133
CD133/2
CD134
CD135
CD136
CD137
CD137L
CD138
CD139
CD14
CD140a
CD140b
CD140b(pY1009)
CD140b(pY1021)
CD140b(pY771)
CD140b(pY857)
CD141
CD142
CD143
CD144
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
CD146
CD147
CD148
CD15
CD150
CD151
CD152
CD153
CD154
CD155
CD156c
CD157
CD158a
CD158a/h
CD158b
CD158b1/b2/j
CD158d
CD158e
CD158e/k
CD158e1
CD158e1/e2
CD158f
CD158g
CD158h
CD158i
CD158j
CD159a
CD159c
CD15s
CD16
CD16/32
CD16/56
CD160
CD161
CD161a
CD162
CD162R
CD163
CD164
CD165
CD166
CD167a
CD168
CD169
CD16b
CD17
CD170
CD171
CD172
CD172a
CD172a/b
CD172b
CD172g
CD173
CD177
CD178
CD178.1
CD179a
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
CD179b
CD18
CD180
CD181
CD182
CD183
CD184
CD185
CD186
CD19
CD191
CD192
CD193
CD194
CD195
CD195(细胞质的)
磷酸化CD195(Ser337)
磷酸化CD195(Ser349)
CD196
CD197
CD198
CD199
CD1a
CD1b
CD1b/c
CD1c
CD1d
CD2
CD20
CD200
CD200R
CD200R3
CD201
CD202b
CD203a
CD203c
CD204
CD205
CD206
CD207
CD208
CD209
CD209b
CD21
CD21/CD35
CD210
CD212
CD213a1
CD213a2
CD217
CD218a
CD22
CD22(pY822)
CD22.2
CD220
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
CD221
CD221(pY1131)
CD222
CD223
CD224
CD226
CD227
CD229
CD229.1
CD23
CD230
CD231
CD233
CD234
CD235a
CD235ab
CD236
CD239
CD24
CD240CE
CD240DCE
CD243
CD244
CD244.1
CD244.2
CD245
CD246
CD247
CD247(pY142)
CD249
CD25
CD252
CD253
CD254
CD255
CD256
CD257
CD258
CD26
CD261
CD262
CD263
CD264
CD265
CD266
CD267
CD268
CD269
CD27
CD270
CD271
CD272
CD273
CD274
CD275
CD276
CD277
CD278
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
CD279
CD28
CD280
CD281
CD282
CD283
CD284
CD284/MD2复合物
CD286
CD289
CD29
CD290
CD294
CD298
CD299
CD2a
CD3
CD3/CD44
CD30
CD300
CD300a
CD300e
CD300f
CD301
CD303
CD303a
CD304
CD305
CD307d
CD309
CD31
CD310
CD312
CD314
CD314(激活)
CD314(封闭)
CD317
CD318
CD319
CD32
CD321
CD323
CD324
CD325
CD326
CD328
CD329
CD32B
CD33
CD334
CD335
CD336
CD337
CD338
CD339
CD34
CD340
CD344
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
CD349
CD35
CD351
CD354
CD357
CD358
CD36
CD360
CD361
CD36L1
CD37
CD38
CD39
CD39L4
CD3D
CD3G
CD4
CD4(结构域1)
CD4(结构域2)
CD4 v4
CD40
CD40bp
CD41
CD41/CD61
CD41a
CD41b
CD42a
CD42b
CD42d
CD43
CD44
CD44(v3)
CD44 (v4)
CD44(v5)
CD44(v6)
CD44(v7)
CD44.2
CD44std
CD44v6
CD44var(v10)
CD44var(v3)
CD44var(v3-v10)
CD44var(v4)
CD44var(v5)
CD44var(v6)
CD44var(v7)
CD44var(v7-v8)
CD45
CD45.1
CD45.2
CD45R
CD45RA
CD45RB
CD45RC
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
CD45RO
CD46
CD47
CD48
CD49a
CD49a/CD29
CD49b
CD49b/CD29
CD49b/CD61
CD49c
CD49d
CD49d/CD29
CD49e
CD49e/CD29
CD49f
CD49f/CD29
CD5
CD5.1
CD5.2
CD5.6
CD50
CD51
CD51/61
CD52
CD53
CD54
CD55
CD56
CD57
CD58
CD59
CD59a
CD6
CD60b
CD61
CD62E
CD62L
CD62P
CD63
CD64
CD64 a,b同种抗原
CD64.1
CD65
CD65s(CD65唾酸)
CD66
CD66a
CD66a/b/c/e
CD66a/c/d
CD66a/c/d/e
CD66a/c/e
CD66a/e
CD66b
CD66c
CD66c/e
CD66e
CD66f
CD68
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
CD69
CD7
CD70
CD70b
CD71
CD72
CD72 a,b,c同种抗原
CD72 b,c同种抗原
CD72.1
CD73
CD74
CD75
CD77
CD78
CD79a
CD79b
CD8
CD80
CD81
CD82
CD83
CD84
CD85
CD85a
CD85d
CD85g
CD85h
CD85j
CD85k
CD86
CD87
CD88
CD89
CD9
CD90.1
CD90.2
CD90.9
CD91
CD93
CD94
CD95
CD96
CD97
CD98
CD98hc
CD99
CD99R
Cdc-123
Cdc-2(p34)
磷酸化Cdc-25A(Ser17)
Cdc-25C
Cdc-37
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
Cdc-45L
Cdc-6
CDc-7
Cdk1
Cdk2
Cdk4
Cdk5
Cdk6
Cdk7
Cdk9
CdkA1
CdkN2A
CdkN3
CDT1
CDX2
CEACAM19
CEACAM20
CEACAM7
CEND1
CENPA
CENPE
CENPF
CENPH
中心体蛋白2
CFAH
cFos
CFTR
CGB5
cGK1
CH2
CHCHD5
CHD3
CHD4
趋化素
CHIPS,C末端
CHIPS,N封端
Chk1
Chk2
硫酸软骨素
CHOP
噬铬粒蛋白C
ChT1
chTOG
cIAP1
cIAP2
CIAS1
CIDEA
CIP4
CISD1
CITED1
CITED2
cJun
磷酸化cJun(Tyr91/Tyr93)
CKMT2
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
CLASP1
网格蛋白
紧密连接蛋白-1
紧密连接蛋白-10
紧密连接蛋白-15
紧密连接蛋白-16
紧密连接蛋白-18(C端)
紧密连接蛋白-18(中部)
紧密连接蛋白-4
紧密连接蛋白-5
紧密连接蛋白-8
CLAW-H
CLEC12A
CLEC1B
CLEC4A
CLEC4M
CLEC9A
CLIP
CLOCK
肉毒梭菌毒素B
CLPP
cMaf
cMet
CMKLR1
CMRF44
CMRF56
cMyb
cMyc
CNDP2
COASY
丝切蛋白
Colec12
胶原蛋白I
胶原蛋白I/III
胶原蛋白II
胶原蛋白III
胶原蛋白IV
胶原蛋白V
胶原蛋白VI
胶原蛋白VII
COMMD1
补体因子B
复合物I免疫捕捉
缀合的胆碱戊二酸
连接蛋白26
连接蛋白30
连接蛋白30.2
连接蛋白30.3
连接蛋白32
连接蛋白36
连接蛋白37
连接蛋白37(C端)
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
连接蛋白37(中部)
连接蛋白39
连接蛋白39(中部)
连接蛋白40(C端)
连接蛋白40(中部)
连接蛋白43
连接蛋白45
连接蛋白45(C端)
连接蛋白46
连接蛋白47
连接蛋白57(C端)
连接蛋白57(中部)
接触蛋白2
COPS3
冠状病毒
冠蛋白1A
冠蛋白1B
皮动蛋白
皮层胸腺细胞
COX I
COX I/III
COX II
COX IV
COX VA
COX VIA1
柯萨奇腺病毒受体
CPF
Cpn10
CPO
CPS1
CPT2
CRABP1
CRABP2
CRALBP
肌酸激酶BB
肌酸激酶MM
CREB
磷酸化CREB(Ser133)
cRel
Cripto1
CRISP3
Crk p38
CrkL
CrkL(pY207)
CROT
CRRY
CRTAM
CRTC3
CRY2
隐花色素I
隐孢子虫
小球隐孢子虫
CRYZL1
CSK
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
CSK结合蛋白
CSPS
cSrc
CST2
CTDSP1
CTNNA3
CTNNBL1
滞蛋白1
滞蛋白2
滞蛋白3
滞蛋白4A
滞蛋白4A/B
滞蛋白4B
皮肤淋巴细胞抗原
CUTL1
CX3CL1
CX3CR1
CXCL1
CXCL10
CXCL13
CXCL9
CXCR7
CXorf26
花青
CYB5R2
CYB5R3
细胞周期蛋白A
细胞周期蛋白A2
细胞周期蛋白B1
细胞周期蛋白B2
细胞周期蛋白D1
细胞周期蛋白D2
细胞周期蛋白D3
细胞周期蛋白E
细胞周期蛋白E2
细胞周期蛋白H
细胞周期蛋白D1/D2/D3
亲环蛋白40
CYLD
CysLT1
血清胱抑素C
血清胱抑素S
细胞色素B245重链
细胞色素B245轻链
细胞色素c
细胞色素P450 17A1
细胞色素P450 19A1
细胞色素P450 1A2
细胞色素P450 2A6
细胞色素P450 2B6
细胞色素P450 2C9
细胞色素P450 2J2
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
细胞色素P450 3A4
细胞色素P450 3A5
细胞色素P450还原酶
细胞角蛋白
细胞角蛋白(酸性)
细胞角蛋白(碱性)
细胞角蛋白(泛反应性)
细胞角蛋白1
细胞角蛋白10
细胞角蛋白10/13
细胞角蛋白13
细胞角蛋白14
细胞角蛋白14/15/16/19
细胞角蛋白15
细胞角蛋白16
细胞角蛋白17
细胞角蛋白18
细胞角蛋白19
细胞角蛋白2
细胞角蛋白20
细胞角蛋白4
细胞角蛋白4/5/6/8/10/13/18
细胞角蛋白40
细胞角蛋白5
细胞角蛋白5/6/18
细胞角蛋白5/8
细胞角蛋白6
细胞角蛋白6a
细胞角蛋白7
细胞角蛋白7/17
细胞角蛋白8
细胞角蛋白8/18/19
D4-GDI
DAB2
DACH1
DAND5
DAP1
DAP12
DAPK1
DAPK2
DARPP32
Daxx
DAZL
DBC1
DCAMKL1
DCC
DCIR2
DCLRE1B
DCP1a
DcR3
DCTN2
DcTRAIL-R1
DcTRAIL-R2
DCXR
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
DDB1
DDDDK标签
DDX3
DDX4
DDX50
DECR1
Dectin1
Dectin2
DEF8
DELETE
delta 1连环蛋白
Delta样蛋白1
Delta样蛋白4
Delta阿片受体
DeltaC
DeltaD
树突状细胞标记物
脱氧胞苷激酶
肌间线蛋白
桥粒核心糖蛋白2
桥粒核心糖蛋白1
桥粒斑蛋白
细胞骨架蛋白
右旋糖苷
DGKA
Dicer
DISC1(C端)
DISC1(中部)
Dishevelled 3
双唾液酸神经节苷脂GD2
双唾液酸神经节苷脂GD3
Dkk1
Dkk3
DLC8
DLK1
Dlx5
DM-GRASP
DMT1
DNA-PKcs
磷酸化DNA-PKcs(Thr2609)
DNAI1
DNAJA2
DNAJB2
DNAJC3
DNAPK
DNM1L
Dnmt1
Dnmt3b
DNP
DOK2
DOK7
多巴胺受体D1
多巴胺受体D3
多巴胺受体D5
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
双皮质素
DP1
DPH2
DPP10
DPP3
DPP9
Dppa4
DPYD
DR3
DRAK1
DRAK2
Drebrin
DTYMK
DUSP23
DUSP27
DUSP3
DUSP5
DUSP6
DUX4
DYKDDDDK表位标签
发动蛋白
发动蛋白1
Dynamitin
动力蛋白轻链2
Dysbindin
Dysferlin
磷酸化Dystroglycan(Tyr893)
E.Coli O/E
E2A-Pbx1
E2F1
E47
E4BP4
结合于I-A的Ea52-68肤
结合于I-A的Ea52-68肤
EAAT1
早期B细胞
EBF1
EBI3
EBP50
ECGF1
ECH1
ECRG4
EDA
EDA-A2R
EDG1
EDG2
EDG3
EDG6
EEA1
EEF1G
EEF2
EEF2K
EEN
EFEMP1
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
EFEMP2
Eg5
磷酸化Eg5(Thr927)
EGF
EGF受体
EGF受体(pY1173)
EGF受体(pY845)
EGF受体(pY992)
EGR1
EGR2
EHD1
eIF1
eIF2C2
EIF2S1
eIF3
eIF3D
eIF3D(p66)
eIF3F
eIF3G
eIF3H(p40)
eIF3I(p36)
eIF3J
eIF3K
eIF4B
eiF4E
eIF4E(pS209)
eIF4E2
eIF5A
eIF6
弹性水解酶
Elk1
Elk1(pS383)
ELK3
延伸蛋白B
延伸蛋白C
EMAP II
Embigin
EMG1
Emi1
EMR3
EMSY
Ena/Vasp-like
EndoG
EndoGlyx-1
假粘蛋白
内皮细胞
内皮脂肪酶
内皮微静脉标记物
内皮
Engrailed1
ENO1
醇化酶1
eNOS
eNOS(pS1177)
Entpd2
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
Eomes
Eos
Epac1
Eph受体A1
Eph受体A2
Eph受体A4
Eph受体B4
Eph受体B6
Ephrin A2
Ephrin A3
EPHX2
EPM2AIP1
EPOR
EPS15R
Epsin 1
Epsin 2
ER-HR3
ER-MP54
ER-TR7
ER81
ERAB
ERCC1
ERG
ERK1
ERK1/2(pT185/pY187)
ERK1/2(pT202/pY204)
ERK1/ERK2
ERK2
ERK5
ERMAP
ERp29
ERp72
红细胞样细胞
Erzin/根蛋白/膜突蛋白
ESAM
雌激素诱导蛋白pS2
雌激素受体
α雌激素相关受体
ETAR
亚乙烯基腺苷
ETS1
EVI2A
EVI2B
EWSR1
EXD1
EXOSC3
EXOSC7
EYA2
EZH1/2
埃兹蛋白
埃兹蛋白(pY353)
F-actin
F10A1
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
F4/80
FAA4
FABP4
因子I
因子IX
因子VIII.vWF(删除)
因子XIIIa
FADD
FAHD2A
FAK
FAK(pS910)
FAM119A
FAM175A
FAM84B
FAM91A1
FANCC
FANCD2
磷酸化范可尼贫血D2(Ser222)
FAP
Fascin
FBP1
FBXO21
FBXO31
FBXO42
FBXO43
Fc受体结合抑制剂
Fc受体IgA+IgM
FcR
FcRL6
FcRLA
FDC
FDFT1
FDPS
FE65
FeLV p27
FEN1
FER
铁蛋白重链
铁蛋白轻链
铁蛋白,线粒体的
FES
胎儿血红蛋白
FGF酸性
FGF碱性
FGF21
FGFR1
FGFR2
FGR
FH
FHL1
纤维蛋白
原纤维蛋白
纤维蛋白原
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
纤维肤A
纤维肤B
成纤维细胞表面蛋白
成纤维细胞/上皮细胞
纤维连接蛋白
纤维连接蛋白受体
纤蛋白5
Ficolin B
丝聚合蛋白
细丝蛋白A
FITC
FITC/俄勒冈绿
FIV
FIV gp120
FIV gp95
FIV p24
FIV p24 gag
FKBP12
FKBP4
FKBP6
FKBPL
FLiC
Flightless1
FLIP
Flt3L
荧光蛋白
FLV gp70
FLYWCH2
FMC7
fMLP受体
FMRP
FNTA
FNTB
卵泡树突状细胞
Fos
FOXA1
FOXA2
FOXC2
FOXD3
FOXI1
FOXJ1
FOXM1
FOXO1
FOXO3A
FOXP1
FOXP3
FPRL1
FR4
Fra2
Fragilis
FRAT1
Frataxin
Frequenin
Frizzled-1
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
FUK
FUS
FXYD3
FYB
Fyn
Fyn(pY528)/c-Src(pY530)
Fyn相关激酶
FZR1
G-CSF
G3BP
G6PD
GAB1
GAB2
GABA B受体2
GABARAP
GAD65
GAD67
GADD34
半乳糖脑苷酯
半乳糖脑苷酯
半乳凝素1
半乳凝素10
半乳凝素3
半乳凝素4
半乳凝素7
半乳凝素8
半乳凝素9
γ突触核蛋白
神经节苷脂GD2
神经节苷脂GD3
神经节苷脂GM1
癌锚蛋白
GAP
GAP43
GAPDH
GARP
GAS2
GAS7
GAT2
GATA1
GATA2
GATA3
GATA4
GATM
GBA3
GBE1
GBP1
GBP2
GBP5
GC1aR
GCDFP15
GCDH
GCK1
GCLM
GCN2
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
GCN5
GCTM2
GDAP1L1
GDF15
凝溶胶蛋白
Gemin1
桥尾蛋白
GFAP
GFP
GILZ
GIMAP4
GIPR
GIT2
GITRL
GLAST
Gli1
胶质纤维酸性蛋白
肠高血糖素
GLIPR1L1
胰高血糖素
糖皮质激素受体
α糖皮质激素受体
葡萄糖1脱氢酶
葡萄糖6磷酸异构酶
GLUH1
GLUT1
GLUT2
GLUT4
GLUT5
谷氨酸受体2
谷氨酸受体2/3
谷氨酸受体3
谷氨酸受体4
谷氨酰胺酶
谷氨酰胺合酶
谷氧还蛋白2
谷胱甘肤NEM
谷胱甘肤NEW
谷胱甘肤过氧化酶1
谷胱甘肤过氧化酶4
谷胱甘肤还原酶
谷胱甘肤合酶
糖元合酶1
糖蛋白IX
糖蛋白VI
GM-CSF
GM130
GM3.2
GNB2
GNB2L1
GNLY
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
GNMT
GnRHR
高尔基蛋白(58K)
高尔基带
GOLM1
GOLPH2
GOSR1
gp340
gp49R
GPA33
GPCR5C
GPR-120
GPR-143
GPR-151
GPR-18
GPR-30
GPR-40
GPR-48
GPR-49
GPR-50
GPR-56
GPR-73A
GPR-73B
GPR-77
GPR-83
GPR-86
GPR-C5C
GPR-C5D
颗粒体蛋白
颗粒溶素
颗粒酶A
颗粒酶B
颗粒酶K
GRAP2
GRASP1
GRASP65
GRB2
GRB7
GRHPR
GRIM19
GRK1
GRK2
GRK3
GRK5
GRK6
生长激素受体
GRP170
GRP94
GSC
GSPT2
GST
GST表位标签
GSTA4
GTF2D1
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
GTPase HRAS
GTPBP4
鸟苷酸激酶
H-2
H-2.m31
H-2Db
H-2Dd
H-2Kd
H2-M
H2-M3
H2A.X
磷酸化H2A.X(Ser139)
H2A1J
H60
HA tag
HADHA
HADHA/HADHB
HADHB
HADHSC
HAND1
HAO1
结合珠蛋白
HARS
HARS2
HBF
HCN4
HDAC1
HDAC10
HDAC2
HDAC3
HDAC4
HDAC6
HDAC9
HDHD1A
HDHD2
HDJ2
HDLBP
HE4
HEC1
HEF1
Helios
造血作用相关巨噬细胞
造血系Cocktail
定向造血干细胞
血红蛋白
血红蛋白F
肝炎B病毒
肝炎B病毒核心抗原
肝炎B病毒E抗原
肝炎B病毒表面抗原(Ad/Ay)
肝炎C病毒
肝炎C病毒核心抗原
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
肝炎C病毒NS4
Hepsin
HER3
HER4
Hes1
己糖激酶
己糖激酶1
己糖激酶2
HFE1
HGF
HGFA抑制剂1
HHEX
HHV8 GPCR
HIBCH
HID1
HIF1AN
HINT1
HIP2
HIPK2
Hippocalcin
组胺H3受体
组织细胞
组蛋白H1
组蛋白H1.0
组蛋白H2A
组蛋白H2B
组蛋白H2B type 1B
组蛋白H3
磷酸化组蛋白H3(Ser10)
磷酸化组蛋白H3(Ser28)
组蛋白H3.3
组蛋白H4
HIV1核心抗原
HIV1 p17
HIV1 p24
HIV1 p55/p17
HIV1 tat
HL60
HLA级别I
HLA-2Kb/2Db
HLA-2kb/2Dd
HLA-A
HLA-A/B/C
HLA-A1/A11/A26
HLA-A1/A36
HLA-A10/A11
HLA-A10/A28/B75
HLA-A10/B62/B71
HLA-A11
HLA-A2
HLA-A2/A25/A32
HLA-A2/A28
HLA-A2/A3/A29
HLA-A2/A69
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
HLA-A2/B17
HLA-A2/B5
HLA-A2/B57
HLA-A23/A24
HLA-A24/A11/A2403
HLA-A25
HLA-A25/A26
HLA-A25/A26/A34
HLA-A25/A32
HLA-A26/A34/B71/B62
HLA-A29
HLA-A3
HLA-A30/A31
HLA-A33/B8
HLA-A34/B71/A26
HLA-A9
HLA-A9/A25/A32
HLA-A9/A32/B13
HLA-B
HLA-B12
HLA-B13/B62/B15
HLA-B14
HLA-B17
HLA-B17/B35/B44
HLA-B21/B70/B55
HLA-B27/B44/B47
HLA-B35/B57/B75/B77
HLA-B44/B75/B17
HLA-B48/B60
HLA-B5/B49/B56
HLA-B7
HLA-B8
HLA-B8/B14
HLA-BC
HLA-Bw4/A9/A32
HLA-Bw6
HLA-Bw6/B77
HLA-级别I自由链
HLA-D
HLA-DM
HLA-DO
HLA-DP
HLA-DQ
HLA-DQ/DR
HLA-DQ1/DQ3
HLA-DQ1/DR7
HLA-DQ3
HLA-DQ6
HLA-DQ7
HLA-DQA1
HLA-DQB1
HLA-DQw1
HLA-DR
HLA-DR/DP
HLA-DR/DP/DQ
HLA-DR1
HLA-DR11
HLA-DR3/DR6
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
HLA-DR4
HLA-DR7
HLA-DR8/DR12
HLA-DR9
HLA-DRA
HLA-E
HLA-G
HLCS
HLF
HLXB9
HMG14
HMG17
HMG4
HMGB1
HMGB2
HMOX1
HMOX2
hnRNPA1
hnRNPC1/C2
hnRNPD
hnRNPK
hnRNPL
hnRNPU
hnRNPUL1
归巢受体
HOXB4
HOXB5
HPa1
HPa2
HPD
HPd1
HPd2
HPi1
HPi2
HPi3
HPi4
HPR1
HPRT1
HPV16 E1/E4
HPx1
HPx2
Hrk
Hsc70
HSD17B1
HSD3B1
HSF1
HSF2
HSF4
HSL
Hsp105
Hsp14
Hsp22
HSP25
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
Hsp27
Hsp40
Hsp47
Hsp60
Hsp70
Hsp70-2
Hsp90
HspA4
HspA6
HSPA9
HspB2
HspB7
HSV标签
HTLV I gp46
HTLV I p19
HtrA2/Omi
人乳头瘤病毒16(E7)
亨廷素
HUS1
Ia(B细胞)
IBA1
IBP2
ICAD
IDO
IFABP
IFNA1
IFNAR1
IFT88
Ig
Ig(多特异性)
IgA
IgA(Fab2)
IgA(H)
IgA1
IgA2
IgD
IgDa
IgDb
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
IgE
IgEa
IgEb
IgG
IgG(Fab H/L)
IgG(Fab)
IgG(Fab2 Fc)
IgG(Fab2 H/L)
IgG(Fab2)
IgG(Fc)
IgG(H/L)
IgG Fd
IgG轻链
IgG/IgM
IgG/IgM/IgA
IgG/IgM/IgA(Fab2 H/L)
IgG/IgM/IgA(Fab2)
IgG/IgM/IgA(H/L)
IgG/IgY
IgG1
IgG1(重链)
IgG1/2a
IgG1/3
IgG1a
IgG1b
IgG2
IgG2/3
IgG2a
IgG2a/b
IgG2b
IgG2c
IgG3
IgG4
IgGDa
IgK
IGKC
IgL
IGLC2
IgM
IgM(Fab2)
IgM(Fc)
IgM(H/L)
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
IgMa
IgMb
IgY
Ihh
Ikaros
IL-10
IL-12
IL-12(p35)
IL-12(p70)
IL-12/IL-23(p40)
IL-13
IL-15
IL-15/IL-15R
IL-16
IL-17D
IL-17A
IL-17A/F
IL-17B
IL-17C
IL-17E
IL-17F
IL-18
IL-18BP
IL-19
IL-1RA
IL-1RN
IL-2
IL-20R2
IL-21
IL-22
IL-23(p19)
IL-23R
IL-24
IL-25
IL-27
IL-27(p28)
IL-28
IL-29
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
IL-3
IL-31
IL-33
IL-34
IL-4
IL-5
IL-6
IL-7
IL-8
IL-9
ILF3
ILK
ILK1
IMP3
输入蛋白9
流感A病毒M2蛋白
流感B病毒核蛋白质
ING1
ING2
ING3
ING4
iNOS
INPP4A
INPP4B
胰岛素
胰岛素降解酶(IDE)
胰岛素受体R
嵌插的DNA
顶体内蛋白
顶体内蛋白
不变NKT
IP10
IQGA1
IRAK1
IRAK3
IRAK4
IRE1
IRF1
IRF3
IRF4
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
IRF5
IRF6
IRF7
IRF7(pS477/pS479)
IRF8
IRF9
IRS1
IRS1(pY896)
IRS2
IRS4
ISG15
ISG20
ISL1
Isthmin1
ITCH
ITK
ITPR1
Jagged2
JAK2
JAK3
JAM2
JAML
日本脑炎病毒NS1糖蛋白
JNK
磷酸化JNK(Thr183/Tyr185)
JNK1/JNK2/JNK3
JNK2
连接粘附分子C
Junctophilin-1(C端)
Junctophilin-1(中部)
Junctophilin-2(C端)
Junctophilin-3(C端)
KAP1
KATNA1
KCNH1
KDEL
KDM4D
Ki-67
KIF22
KIF3A
KIF4A
KIFA3
整合素2
动基体类膜蛋白11(KMP-1))
KIR-2.1
KIR-2D(泛CD158)
KLF4
KLF6
KLH
KLHL11
KLRA3
KLRC1
KLRG1
KMT4
KMT5A
KOR-SA3544
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
KS1/4
Ksp37
KSR1
Ku70
Ku70/80
Ku80
Kudoa Thyrsites
Kunitz蛋白酶抑制剂
Kv4.2
L/S-MAG
标记检测试剂
乳酸脱氢酶
乳酸脱氢酶B
Lambda
核纤层蛋白A
核纤层蛋白A/C
核纤层蛋白B受体
核纤层蛋白B1
核纤层蛋白B2
核纤层蛋白C
层粘连蛋白
层粘连蛋白5
层粘连蛋白受体
LAMP2a
LAMP2b
LAT
LAT(pY171)
LAT(pY226)
LBP
LC3
LC3B
LCAT
Lck
Lck(pY505)
LDH1
LDH1/B/C
LDL(MDA氧化)
LDLR
LEF1
利什曼虫LPG(重复表位)
利什曼虫主要蛋白抑制酶(GP-63)
LEKTI
白血病抑制因子
白三烯A4水解酶
白三烯B4受体
LHX3
LI-钙粘蛋白
LIF
DNA连接酶I
DNA连接酶III
LIM激酶2
LIME1
LIMK1
LIMS1
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
Lin28
Lineage Cocktail
Lipin 1
LIS1
肝脏羧酸酯酶1
LKB1
LMO2
LOX
LOX1
LRP5/6
LRP6
LRPAP1
LSD1
LSP1
LSS
萤火素酶
Ly-108
Ly-49A
Ly-49A/D
Ly-49AB6
Ly-49C/F/I/H
Ly-49C/I
Ly-49D
Ly-49E/F
Ly-49F
Ly-49G
Ly-49G2
Ly-49G2B6
Ly-49H
Ly-49I
Ly-51
Ly-6A.2/Ly-6E.1
Ly-6A/E
Ly-6b
Ly-6B.2
Ly-6C
Ly-6D
Ly-6G
Ly-6G/C
Ly-6K
Ly-77
Lyn
LYRIC
溶血磷脂酶1
溶酶体酸脂酶
溶菌酶
溶菌酶
Lyve1
M-CSF
M13噬菌体外壳蛋白g8p
M13噬菌体蛋白
MAA
Mac-2BP
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
macroH2A.1
巨噬细胞
巨噬细胞激活蛋白
巨噬细胞半乳糖凝集素
巨噬细胞/粒细胞
巨噬细胞/单核细胞
MAD2
MadCAM1
MADD
MADH7
MAFB
MAG
MAGE-A
MAGE1
MAIR2
MAIR4
MALT1
乳球蛋白A
MAP1LC3A
MAP2
MAP2B
MAP2K1IP1
MAP3K8
磷酸化MAP4(Ser768)
MAP4K1
MAP4K4
MAPK12
MAPK6
MAPKAP激酶2
磷酸化MAPKAP激酶2(Thr334)
MARCKS
MARCO
缘区B细胞
MARK2
MARK3
MART1
肥大细胞
肥大细胞蛋白酶11
成熟巨噬细胞标记物
MBD1
MBD2
MBL
MCL1
MCM2
MCM3
MCM4
MCM5
MCM6
MCM7
MCP-1
MCP-4
MCP-8
MCSF
MD1
MD2
MDC
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
MECT1
MEF2A
MEIS1
MEK1
MEK1(p298)
MEK1(pS218)/MEK2(pS222)
MEK1/2(pS222)
MEK2
MEK3
MEK4
MEK5
MEK6
MEK7
MEKK1
MEKK2
MEKK3
MEKK4
黑素瘤
MELK
MEMO1
Mena
Menin
MEOX2
Merlin
MERTK
间皮蛋白
金属硫蛋白
MetRS
mGluR5
MGMT
MHC级别I
MHC级别I(H-2Db)
MHC级别I(H-2Dd)
MHC级别I(H-2Dk)
MHC级别I(H-2Dq/Lq)
MHC级别I(H-2Kb)
MHC级别I(H-2Kb/Db)
MHC级别I(H-2Kb/Dd)
MHC级别I(H-2Kd a3结构域)
MHC级别I(H-2Kd)
MHC级别I(H-2Kd/Dd)
MHC级别I(H-2Kd/Dd/q/u/v)
MHC级别I(H-2Kk)
MHC级别I(H-2Kq)
MHC级别I(H-2Ks)
MHC级别I(H-2Ld)
MHC级别I(H-2Ld/Db)
MHC级别Ib(H2-M3)
MHC级别II
MHC级别II(DQ)
MHC级别II(DR)
MHC级别II(I-A)
MHC级别II(I-A/E)
MHC级别II(I-Ab)
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
MHC级别II(I-Ab/Ad)
MHC级别II(I-Ab/As)
MHC级别II(I-Ad)
MHC级别II(I-Ak)
MHC级别II(I-Ak/Ad/Ab/Aq/Ar)
MHC级别II(I-Ak/As)
MHC级别II(I-Ap)
MHC级别II(I-Aq)
MHC级别II(I-E)
MHC级别II(RT1B)
MHC级别II(RT1Bu)
MHC级别II(RT1D)
MHC Qa1b
MICA
MICA/MICB
MICB
微褶(M)细胞
微管相关蛋白2ab
微管相关蛋白RP/EB 2
Midkine
盐皮质激素受体
MIPEP
线粒体
Mitofilin
线粒体融合蛋白1
线粒体融合蛋白2
有丝分裂细胞
MKK6
MLH1
MLK3
MLL1
MLLT11
MMP1
MMP10
MMP11
MMP12
MMP13
MMP14
MMP15
MMP17
MMP19
MMP2
MMP20
MMP21
MMP26
MMP3
MMP8
MMP9
Mnk1
mNOS
MnSOD
膜突蛋白
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
单胺氧化酶B
单核细胞/粒细胞
单核吞噬细胞
小鼠胚胎成纤维细胞(mEF)饲养细胞
小鼠谱系
MPP1
MRCL3
MRE11
MRGPR-X2
MRI1
MRP14
MRP2
MRP3
MRP4
MRP5
MRP6
MRP8
MRP8/14
MSC(W8B2)
MSC(W3D5)
MSC(W5C5)
MSC(W7C6)
MSC/NPC
MSH2
MSH6
MSI2H
MSK1
MST1
MST1/MST2
MST3
MST4
MST4/MST3/STK25
mTOR
Muc-16
Muc-2
Muc-3
Muc-4
Muc-7
MULT-1
Munc13-4
Munc18
MUPP1
Mus81
Musashi1
蕈毒碱乙酰胆碱受体2
肌肉肌动蛋白
肌肉结合样1
MVP
MYBBP1A
MYBPC3
Myc tag
MyD88
髓磷脂碱性蛋白
髓鞘少突胶质细胞糖蛋白
髓磷脂PLP
骨髓抗原
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
髓细胞核分化抗原
髓系
Myocilin
肌细胞生成素
肌凝蛋白重链
肌凝蛋白IIA
肌球蛋白轻链2
肌球蛋白轻链3
肌球蛋白轻链激酶
肌凝蛋白磷酸酶
肌凝蛋白磷酸酶1/2
MYST2
NADH2
Naf1
NAK
Nanog
NAPE-PLD
NAT1
天然脂壁酸
利钠肤受体A
自然杀伤细胞
自然杀伤细胞激活结构
NBS1
NC1.1
NCF4
Nck
NCOA1
NCOA2
NCX1
NDUFAF1
NDUFB4
NDUFS3
NEDD8
NEK2
NEK6
NEK7
NEK9
磷酸化NEK9(Thr210)
巢蛋白
NETO2
Neurabin1
神经调节蛋白1
神经调节蛋白3
神经母细胞瘤
神经D1
神经D2
神经纤维瘤蛋白
高分子量神经丝蛋白
中等分子量神经丝蛋白
神经原素2
神经激肤1受体
神经元特异性醇化酶
神经元生长因子受体
神经紧张素受体1
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
NFATc1
NG2
NGF
Nhedc2
NHERF1
Nicastrin
Ninein
硝基酪氨酸
NKG2A/C/E
NKG2AB6
NKp80
NKX3.1
NM23A
NMDA受体2A
NMDA受体2B
NMDE2
NMDZ1
NMNA2
nMyc
nNOS
NNTM
痛敏肤
Nod2
Nodal
Noggin
NONO
非特异性细胞毒性细胞
Notch1
Notch2
Notch3
Notch4
NOX2
NOX4
NOXA2
NPC
NPM-ALK
磷酸化NPM/B23(Thr199)
磷酸化NPM/B23(Thr234/Thr237)
NPY5R
NQO1
NR2E1
NRC2C
Nrf2
NRG3
NSPA/B
NTAL
NTF97
核仁素
磷酸化核仁素(Thr76/Thr84)
核基质蛋白
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
NUDC
NUMA1
Nur77
O乙酰基GD3
Oct2
Oct3/4
Oct3/4A
Oct4
ODAG
OGDH
OLIG1
OLIG2
少突胶质细胞标记物
少突胶质细胞标记物O1
少突胶质细胞标记物O4
制瘤素M受体
Orai1
OSCAR
OSR1
骨连结蛋白
骨桥蛋白
骨保护素
Otx2
OVA(SIINFEKL)H-2Kb
卵巢细胞标记物
卵清蛋白
卵巢癌相关抗原
OX-62
p120连环蛋白
p120连环蛋白(pS268)
p120连环蛋白(pS288)
p120连环蛋白(pS879)
p120连环蛋白(pT310)
p120连环蛋白(pT916)
p120连环蛋白(pY228)
p13
p130
p130 Cas
p130 Cas(pY249)
p14ARF
p150,95
p19ARF
p21
p22phox
p23
p27Kip1
P2RX4
P2RY8
P2X3
P2X7
P2Y6
p34Cdc-2
p38
p38 MAPK(pT180/pY182)
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
p400
p53
乙酰化p53(Lys305)
乙酰化p53(Lys382)
磷酸化p53(Ser15)
磷酸化p53(Ser37)
磷酸化p53(Ser392)
p53BP1(Ser1778)
p57Kip2
p60 CAF1
p62
p63
p63(TA)
p90 Rsk
磷酸化p90 Rsk(Thr368/Ser372)
p95 NBS1
p97
PABP1
PABP2
PABPN1
PAC1
PAD2
PAG1
PAK1
PAK2
PAK3
泛肌动蛋白
泛巨噬细胞
泛内皮细胞抗原
PAR1
副流感病毒类型1
副流感病毒类型2
副流感病毒类型3
PARC
PARD3
PARK7/DJ1
PARP,裂解形式
PARP16
PARP4
PARVA
Pax2
Pax5
Pax6
Pax7
Pax8
Pax9
桩蛋白
磷酸化桩蛋白(Tyr118)
磷酸化桩蛋白(Tyr31)
PBEF
PBK
PBP
PBR
PBX3
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
PCB
PCNA
PCYT1A
PD-1H
PD-ECGF
PDC-TREM
PDCD4
PDCD6
PDE3B
PDECGF
PDGF-AA
PDI
PDK1
PDK2
PDPK1
PDPK1(pS241)
PDX1
PDZK1
PE
PECR
PEI-转铁球蛋白
Pellino 1
凝集素3
PEPD
穿孔素
过氧化物氧化还原酶1
过氧化物氧化还原酶2
过氧化物氧化还原酶6
PEX5
PF4
PGIS
PGP9.5
PGRP-Ia
PGRP-S
PHD1
PHD2
磷脂酰丝氨酸
磷酸化SHIP
磷脂酶A2激活蛋白(PLAP)
磷脂酶D1
磷丝氨酸/苏氨酸/酪氨酸
磷酸酪氨酸
PI 3激酶p150
PIAS1
PIAS3
PICK1
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
PIM1
PIM2
Pin1
PINK1
PIR-A/B
Pirh2
PIST
PiTX3
PIWIL2
PKAR2
PKC
PKCq
PKN
PKN2
PKR
PKX1
PLA2G1B
胎盘碱性磷酸酶
胎盘蛋白14
Plakophilin 3
网素L
血小板
PLAU
网蛋白
多效生长因子
神经丛蛋白A1
神经丛蛋白B2
PLGF
PLK1
磷酸化PLK1(Thr210)
PLK4
PLSCR1
PLVAP
PLZF
PMCA(1-4)
PMCA4
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
PMEL17/SILV
PMN
PMP70
PMS2
PNAd
PNPH
足糖萼蛋白
平足蛋白
POKEMON
多组氨酸标签
PON1
PON3
PPM1A
PPP1A
PPP5C
PPP6C
PR3
PRA1
PRC1
前BCR
前清蛋白
早衰蛋白1
早衰蛋白2
朊蛋白PrP
PRKRA
PRLR
PRMT1
PRMT5
原松弛素1/2
原松弛素2
抑制蛋白1
黄体酮受体
抗增殖蛋白
前动力蛋白1
前动力蛋白2
催乳素
ProMBP1
前列腺素D2受体
前列腺素脱氢酶1
前列腺素E受体EP3
前列腺细胞表面抗原
前列腺特异性抗原
前列腺酸性磷酸酶
蛋白酶体20S C2
A蛋白
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
G蛋白
蛋白激酶D2
蛋白磷酸酶抑制剂1
S蛋白
蛋白酶激活受体4
凝血素
PSA-NCAM
PSD95
绿脓杆菌
PSMA
PSMD14
牛皮癣素
PTAFR
PTBP1
PTEN
PTGER2
PTGER4
PTHLH
PTK7
PTP1B
PTP4A2
PTPS
PTRH2
PU.1
PU60
PUMA
山松醇1
山松醇2
PXR
PYCARD
Pygopus2
Pyk2
Pyk2(pY402)
丙酮酸脱氢酶E2
丙酮酸脱氢酶E2/E3bp
q2
Qa1(b)
Qa2
RAB11A
RAB25
RAB27A
RAB4
RAB5a
RAB9
Rac1
Rac1/Cdc42
RAD17
磷酸化RAD17(Ser645)
RAD23A
RAD51
RAD54
RAD9A
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
根蛋白
RAF1
RAGE
RAIDD
虹鳟鱼Ig
Ra1BP1
RanBP9
RanGAP1
RAP1A/RAP1B
RAP1GAP
Raptor
RAS
RASGAP
RASGRF1
RASSF1A
Rb
Rb(a.a.332-344)
Rb(pS780)
Rb(pS807/pS811)
RbAp46
RbAp48
RBC
RBC(多克隆兔)
RBM35A
RBP4
RBX1
RCC1
RcRL6
红细胞
松弛素1
松弛素1/2
松弛素2
RelB
RELT
肾素
RENT1
Reptin
排斥导向分子C
抵抗素
REST
Ret
成纤维网状细胞和网状纤维
Reticulon1A
网状细胞
视网膜母细胞瘤1
RFLAT1
RFP
RGS6
RGS7
RGS9
RHEB
Rho
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
RhoA
RHOC
RhoGAP
RhoGDI
RIAM
RICTOR
RIG1
RIP1
RIP2
利妥昔单抗
RLA DQ
RLA DR
RNA聚合酶II
RNA聚合酶II CTD重复YSPTSPS
RNASE-L
RNASE1
RNF144B
RNF168
RNF36
RNPEP
ROCK1
ROR1
ROR2
ROS
RPA32/RPA2
RPA70
RPS6
RSF1
RSK1 p90
RSK2
RSK3
RSK4
RT1A
RT1Aa
RT1Aa,b
RT1Aa,b,1
RT1Ac
RT1Au
RT1B
RT6.1
RT6.2
兰尼碱受体
RYK
RyR
S-Tag
S100A1
S100A10
S100A13
S100A4
S100A6
S100A9
S6(pS235/pS236)
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
S6(pS240)
S6(pS244)
S6K
SAA4
Sall4
副伤寒沙门氏菌A
鼠伤寒沙门氏杆菌
鲑科Ig(H和L链)
鲑科Ig(H链)
SAM68
SAMD2
SAP
SARA
SATB1
SATB2
SC5A5
SC6A4
SCAI
SCD1
拼接酶1
SCY1样3
SDF1
SDHA
SDHB
分泌组分
Securin
SELP
Sema4A
Sema7A
SENP1
SEPP1
SERCA2
丝氨酸蛋白酶抑制蛋白B1
丝氨酸蛋白酶抑制蛋白B2
丝氨酸蛋白酶抑制蛋白B6
Sestrin1
SFRP2
SGK1
SHC1
波伊德氏志贺氏菌
SHIP1
SHP1
SHP2
SHP2(pY542)
SIAH2
SIGIRR
Siglec-10
Siglec-8
Siglec-9
Siglec-F
Siglec-H
SIK2
SIRT1
SIRT2
SIRT3
SIRT5
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
SIT1
SIX2
SKP1A
SLA-DR
Slan
SLC1A3
SLC1A7
SLC22A1
SLC22A5
SLC26A6
SLC26A7
SLC30A4
SLC39A11
SLC4A3
SLC6A19
SLC6A6
SLC7A10
SLC7A14
SLC7A3
SLC7A8
SLC8A2
SLC9A6
SLP76
SLP76(pY128)
SMAC
SMAC3
SMAD1
SMAD1(pS463/465)
SMAD1/5
SMAD1/9
SMAD2
SMAD2/3(pS465/467)DELETE
SMAD3
SMAD4
SMAD5
SMAD6
SMC1
SMC1L1
SMN
平滑肌特异性蛋白
SMURF2
SNAP25
SNX1
SOAT1
SOCS1
SOCS2
SOCS3
SOCS6
SOD2
钠钾ATP酶
音猬因子
Sortilin
SOSC3
SOX1
SOX10
SOX17
SOX18
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
SOX2
SOX2(COOH末端)
SOX2(NH2末端)
SOX9
SP-D
Sp1
Sp3
SPHK1
Spt16
Src(pY418)
SREBP1
ssDNA
SSEA3
SSEA4
SSEA5
SSH3BP1
SSR2
SSR5
SSRP1
SSX2IP
Stat1
Stat1(N末端)
Stat1(pS727)
Stat1(pY701)
Stat2
Stat3
Stat3(pS727)
Stat3(pY705)
Stat4
Stat4(pY693)
Stat5
Stat5(pY694)
Stat5a
Stat5b
Stat6
Stat6(pY641)
磷酸化Stathmin/Op18(Ser16)
Stathmin1
Stefin B
干细胞因子
STIM1
STK3
STK33
STK39
STOM
STRO1
STUB1
SULT1A1
SULT1A3/SULT1A4
SULT1C2
SULT2A1
SUMO1
SUMO2
SUMO3
SUN1
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
融合抑制蛋白
SUPT16H
生存蛋白
磷酸化生存蛋白(Thr34)
SV40大T和小t抗原
SWC1a
SWC6
SYBL1
Syk
Syk(pY348)
突触蛋白I
突触蛋白II
Synaptojanin2
突触素
多配体蛋白聚糖4
SynGAP
Synip
突触融合蛋白
突触融合蛋白6
Syntrophin
SYWC
T细胞(泛反应性)
T淋巴细胞
T细胞和B细胞激活抗原
T7 tag
TAB1
TACE
TACI
TAF172
TAF250
TAG72
Talin1
Talin2
Tamm Horsfall(尿类黏蛋白)
TANK1
TAP1
TAP2
TARDBP
TARP
抗酒石酸盐酸性磷酸酶
TAS1R1
Tau
TBA1B
Tbet
TBK1(pS172)
TBX1
TC10
TCF3
TCF7L1
TCF7L2
TCL1
TCR
TCR DO11.10
TCR HY
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
TCTP
TdT
Tec
TEF1
TEM8
肌腱蛋白C
TER119
TERF2
末端脱氧核苷酸转移酶
TERT
四连接素
TFF3
TFIIB
TGN38
TGN46
THAP11
THEMIS
硫氧还原蛋白
硫氧还原蛋白还原酶1
ThPOK
凝血酶受体
血小板
凝血酶致敏蛋白
胸苷激酶1
甲状腺球蛋白
TIA-1
TIAM2
Tie1
Tie2(pY1102)
Tie2(pY992)
TIGIT
Tim1
Tim2
Tim3
Tim3 Fc融合蛋白
Tim4
Tim50
Timeless
TIMP1
TIMP2
TIP49A
TIRAP
TIS11b
TL1A
TLK1
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
TLR11
TLR12
CD285
TLR7
TLR8
TMEFF2
TMPS2
TMSA
TMTSP
TNAP
TNAP3
TNFR相关蛋白
TNPO3
Tollip
TOMM20
TOMM22
TOP1
TOP2A
TOP2B
TORC2
Torsin A
TOX
TPH1
TPPP
TPTE
TR11B
TRA-1-60
TRA-1-60R
TRA-1-81
TRA-2-49
TRA-2-54
TRADD
TRAF2
TRAF4
TRAF5
TRAF6
TRAM2
转铁蛋白
谷氨酰胺转移酶
谷氨酰胺转移酶2
转酮酶
TRAP1
TRAPPC2
Trem-like 2
Trem-like 4
TRIB2
TRIB3
TRIM
TRIM25
TRIM29
TRK
TrkA
TrkC
Trop2
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
原肌球蛋白1
TROY
TRPC6
TRPM2
TRPM8
TRX1
布氏锥虫主要溶酶体蛋白
布氏锥虫原周期蛋白(EP)
刚果锥虫原周期蛋白(procyclin)
克氏锥虫LPG
磷酸化TSC2(Ser664)
磷酸化TSC2(Thr1462)
TSG101
TSHR
TSLP
TSLP受体
TSPO
TTF1
Tubb3
结节蛋白
上皮来源的肿瘤抗原
Twist2
TXNIP
TYK2
TYMS
Tyro3
酪氨酸酶
酪氨酸羟化酶
UACA
UBA52
UBC9
UBE2
UBE2L3
UBE2L6
UBE2M
UBE2N
UBF
UBF1
泛素
UBK63
UCH37
UCK
UCP2
UCP3
UFM1
ULBP1
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
ULBP2
ULBP4
ULK3
UNC5A
UNC5B
UNG
uPA
UQCRC1
UQCRC2
Urm1
URP2
USF1
USP11
USP13
USP22
USP28
USP7
UTF1
V5标签
VAMP5/8
VAP1
VASA
VASP
VAV1
VAV2
VAV3
VDAC1
VEGF
VEGF-120
VEGF-A
VEGF-R1
VELIS-3
VGLU1
绒毛蛋白
波形蛋白
粘着斑蛋白
Viperin
VIPR1
维生素D结合蛋白
维生素D受体
玻璃粘连蛋白
VMAT2
vMyb/cMyb
von Willebrands因子
VRK1
VSV-G标签
WAPL
WASP
WC14
WC15
wCD44
WIP(pS488)
WNT1
WNT16
WNT2
WNT5B
WNT6
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
WSTF
WWOX
黄嘌呤氧化酶
XBP1
XBP1(COOH末端)
XBPs
XCL1
XIAP
XPC
XPNPEP3
XRCC2
XTP4
YAP1
YB1
YES1
YY1
ZAP-70
ZAP-70(pY292)
ZAP-70(pY319)
ZAP-70(pY319)/Syk(pY352)
ZBP-1
ZIPK
ZO-1(中部)
ZONAB(中部)
斑联蛋白
IL-33R
Globo H
CCL8
Siglec-G
CD307e
CLEC6
Snaill
SMAD1(pS463/pS465)/SMAD8(pS465/pS467)
SMAD2(pS465/pS467)/SMAD3(pS423/pS425)
NKX6.1
FAK(pY397)
Btk(pY223)/Itk(pY180)
ERK3
MCP-3
CD238
β2微球蛋白[b,c]
核干细胞因子
GPR-49(中间LRR)
GPR-49(N-末端)
螺旋蛋白
三硝基酚
膜联蛋白VII
CD301a
CD301b
mTOR(pS2448)
PI16
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
MSC(W5C5)
LAMP5
GPR-19
FPRL2
CXCL5
PAR2
ULBP6
ULBP2/5/6
IL-17B受体
ULBP3
精氨酸酶1
碱性磷酸酶
ULBP3
TrkB
骨钙蛋白
APJ
FGFR3
SR-A1
Rae-1(pan)
CXCL12
TREM2
鼠短尾突变体表型
CLEC5A
Mer
XCR1
AML2
von Willebrands因子A2
MMP7
GLP-1R
FR1
IL-1RAcP
紧密连接蛋白-6
瘦素受体
Caherin 6
IL-1R II型
结合素4
Delta样蛋白3
ChemR23
GPR-39
CD158b2
LRIG1
神经菌毛素2
GPR-44
Eph受体B2
磷脂酰肌醇蛋白聚糖3
IL-17C受体
BMPR1B
IL-31RA
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
OCIL
Frizzled-7
IL-26
GPR-15
神经丛蛋白D1
CD158
FPR1
HBEGF
维生素D3
神经丛蛋白B1
生长抑素受体2
OV-6
CXCL16
Siglec-E
EDG5
Ninjurin-1
MHC级别II(I-Ed/j/k/p/r/u/v)
ThB
MAP-2(2a&2b)
MHC级别I(H-2b/p)
MHC级别I(H-2s/p/q/d/u/r)
MHC级别I(H-2s/f)
CDw60
Bad Phospho(Ser112)
裂解的半胱天冬酶3(Asp175)
磷酸化Chk1(Ser345)
磷酸化Chk2(Thr68)
磷酸化细胞周期蛋白D1(Thr286)
磷酸化cFos(Ser32)
FosB
乙酰化组蛋白H3(Lys9)
磷酸化HS1(Tyr397)
磷酸化Hsp27(Ser82)
ID3
磷酸化-IRAK4(Thr345/Ser346)
磷酸化-cJun(Ser73)
S6(pS240/pS244)
Syk(pY525/pY526)
C23
p27
磷酸化cJun(Ser63)
ENPP1
Twist1
钙粘蛋白M
CD302
CD66d
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
CLEC14A
CD242
多配体蛋白聚糖2
CDO
Cryptic
内皮缩血管肤B受体
FR3
IGSF3
CD85f
间质蛋白酶
MCEMP1
mGluR4
Stabilin1
Stabilin2
钙粘蛋白13
GPR-109A
TSPAN8
Reg1A
钙粘蛋白12
ECE1
FABP5
IGSF4C
Trem-like 1
激活素A受体IIA型
ALK7
BCAM
BLAME
CEACAM4
紧密连接蛋白-3
CLP24
CRHR1
DC-STAMP
Eph受体B3
FATP4
FcRL1
FcRL2
FcRL3
FSH-R
Gi24
组胺H1受体
Neu5Gc
Lin28A
ATM(pSer1981)
CD158k
KOR
CD85i
LRIG3
LRP4
MMP16
MS4A4A
NAALADase-like 2
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
1型神经肤Y受体
MS4A3
PEAR1
PEDF受体
神经丛蛋白A4
原钙粘附蛋白1
ROBO2
ROBO4
EDG8
Scavenger受体A5
Semaphorin 4A
Semaphorin 4B
Semaphorin 6A
Siglec-16
生长抑素受体3
STING
GPBAR1
TM4SF4
TMEM87A
TSPAN2
VEGF-R1,2,3
ADAM15
钙网蛋白2
补体因子H-相关4
CXCL6
CD158a/h/b2/f/g
结合于I-Ab的Ea52-68肤
HLA-Bw4
磷酸化ATF1(Ser63)
表皮调节素
FATP1
纤维调节素
弗林蛋白酶
甘丙肤
IL-11
CD306
MFG-E8
MINA
Oct4A
OLIG1,2,3
制瘤素M
Semaphorin 3E
Slug
SOX3
STYK1
LTBP1
TIMP3
VAP-B
WNT9a
5HT2C
AATK
ACLP
ADAMTS15
α1B肾上腺素受体
APLP1
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
荧光素/俄勒冈绿
L3MBTL3
CCL1
PRDM4
ACTH
PDZ结合激酶
HuC/HuD神经元蛋白
TDRD3
EP300
碳酸酐酶VI
胆囊收缩素A受体
CCL23
CD1e
软骨凝集蛋白
腱蛋白样2
紧密连接蛋白-10b
紧密连接蛋白-11
紧密连接蛋白-12
紧密连接蛋白-17
CLEC2A
凝血因子VII
CXCL1/2/3
DDR2
DPCR1
二肤基肤酶6
上皮膜蛋白3
Endoglycan
颗粒体蛋白C
FATP2
FATP5
FcRLB
GLP-2R
GLUT3
磷脂酰肌醇蛋白聚糖6
GPR-22
GPR-37
GPR-37L1
INSRR
LINGO1
LINGO2
mGluR2
mGluR7
MMP25
神经介素B受体
NRAGE
骨激活素
Porimin
前动力蛋白受体1
Prominin2
Semaphorin 3A
SLAP-130
生长抑素受体5
SCARF1
STAMP2
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
TAFA3
TAFA4
TM4SF18
结节性硬化1
TCF8
CMG2
IL-17D受体
巨噬细胞刺激蛋白受体
Siglec-11
多配体蛋白聚糖3
CD85e
SOX7
激活素A受体IA型
碳水化合物磺基转移酶15
CD300b
CELSR3
凝血因子II
DC-SCRIPT
DSCAM-L1
FLRT1
Frizzled-6
磷脂酰肌醇蛋白聚糖1
IGSF4B
IL-1R9
BAZ2B
BRD4
Kell
Kremen2
LAX1
CD85c
MIF
脑啡肤酶2
OBCAM
神经丛蛋白C1
RGM-B
Xg
DCBLD2
ASAM
桥粒糖蛋白1
Frizzled-3
MMP24
TOR
WNT3a
磷脂酰肌醇蛋白聚糖5
Jagged1/Jagged2
Pax3
CELSR2
细胞周期蛋白D1/D2
神经丛蛋白A2
TAFA5
FR4
CD315
NKG2I
RAMP2
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
TNFRH3
蛋白生物素
GPVI
MS4A4B
PIR-B
Semaphorin 4F
IL-1F6
CD39L3
接触蛋白3
CLEC4B
MC3R
PGRP-L
PLET1
ADAM9
AMIGO3
CD99-L2
Eph受体A5
Ephrin B2
CD316
Kremen1
Eph受体B1
神经丛蛋白B3
DMBT1
FcRn
LIMPII
MUCDHL
Patched1
SLC39A4
IGSF4A
PRAT4B
HHV8-ORF74
磷酸化4E-BP1(Thr36/45)
磷酸化4E-BP1(Thr69)
DCAR1
Von Hippel-Lindau
同型对照
颗粒酶M
REA同型对照
CD300LG
MR1
CD327
B7-H6
CLEC4G
BATF3
IL-38
单羧酸转运蛋白1
MC5R
TCF7
TM4SF1
GPR-49(CRL区)
CD156a
ADAM33
ADAMTS13
CCL16
CXCL17
Deltex1
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
FBXO15
GPR34
GPRC5A
胰岛素原
JAK1
MEP1A
下视丘分泌素受体2
p70S6K
STRA6
胰岛素R/IGF-I R异四聚体
SPARCL1
Spi-B
TRAM
羧肤酶E
胰岛细胞自身抗原1
Patched2
ST8SIA2
AML1(pS249)
BRF1/2
磷酸化组蛋白H3(Thr11)
MEK1(pT286)
MMP16
磷酸化MNK(T197/T202)
NUMB
磷酸化Hsp27(Ser78)
SIRT1(pS47)
ZAP-70(pY493)
ZAP-70(pY315/pY319)
sRAGE
mCherry
TIMP4
SRC
ZAP-70(pT493)
磷酸化TSC2(S939)
RagC
SHIP2
MKK4(pS257)
CD79a(pY182)
TRAF1
EVI1
SRC3
SOX11
IL-17F同型二聚体
CCRL1
FOXP2
IFNAR2
REA对照
CD228
Muc-13
P2X7R
Btk(pY223/Itk(pY180)
表8.与本申请描述的水凝胶颗粒一起使用的细胞表面标记物。
CD248
GILT
恢复蛋白
心肌肌钙蛋白I
NKX2.2
HLA-B7/B27
肌球蛋白轻链2a
肌球蛋白轻链2v
上皮细胞抗原
CD92
在一种实施方式中,水凝胶颗粒的群体用于确定在靶细胞群落上检测特定细胞表面标记物或其组合的动态量程和/或灵敏度。例如,可以调节水凝胶颗粒的群落以使其具有靶细胞的SSC和/或FSC波形,且将水凝胶颗粒的子群落用特定数目的细胞表面标记物例如细胞表面受体或其结构域如其表面抗原结合区的拷贝衍生化。例如,可以将水凝胶颗粒的个体子群落各自衍生化以具有独特数目的拷贝,例如,一个子群落将包含100份拷贝的细胞表面标记物,第二子群落将包含1,000份拷贝的同一细胞表面标记物,第三子群落将包含10,000份拷贝的同一细胞表面标记物等。将水凝胶颗粒的群落的各细胞表面标记物荧光染色,并检测各子群落中的水凝胶颗粒。鉴于此,对于具有各细胞标记物的靶细胞,水凝胶颗粒的子群落可用于产生荧光发射的标准曲线。细胞表面标记物可以是其提供的任何细胞表面标记物,或其结合区,或本领域技术人员已知的细胞表面标记物。
本申请的水凝胶颗粒在例如染色和通过流式细胞计数法或FACS进行分析等过程中的行为相似于靶细胞。例如,在一种实施方式中,水凝胶颗粒具有的一种或多种光学性质基本上类似于表1、表2或表3中陈列的细胞类型之一。
在一些实施方式中,靶细胞是免疫细胞。免疫细胞的非限制性实例包括B淋巴细胞,也称为B细胞,T淋巴细胞,也称为T细胞,自然杀伤(NK)细胞,淋巴因子激活的杀伤(LAK)细胞,单核细胞,巨噬细胞,嗜中性粒细胞,粒细胞,肥大细胞,血小板,朗格汉斯细胞,干细胞,树突状细胞,外周血单核细胞,肿瘤浸润(TIL)细胞,基因修饰的免疫细胞,包括杂交瘤,药物修饰的免疫细胞,以及本申请列出的任何细胞类型的衍生物、前体或祖细胞。
在一些实施方式中,靶细胞包括具有共享属性的特定类别细胞的所有细胞。例如,靶细胞可以是淋巴细胞,包括NK细胞、T细胞、和B细胞。靶细胞可以是激活的淋巴细胞。
在一些实施方式中,靶细胞是原代细胞,培养细胞,建成细胞,正常细胞,转化细胞,受感染细胞,稳定转染的细胞,瞬时转染的细胞,增殖细胞,或分化终末细胞。
在一种实施方式中,靶细胞是原代神经元细胞。多种神经元均可以是靶细胞。作为非限制性实例,靶细胞可以是原代神经元;建成神经元;转化神经元;稳定转染的神经元;或运动或感觉神经元。
在其它实施方式中,靶细胞选自:原发性淋巴细胞,单核细胞,和粒细胞。
靶细胞本质上可以是任何类型的细胞,包括原核细胞和真核细胞。
适宜的原核靶细胞包括但不限于,细菌例如大肠杆菌(E.coli),各种芽孢杆菌属,和极端微生物细菌例如嗜热生物。
适宜的真核靶细胞包括但不限于,真菌例如酵母和丝状真菌,包括以下属种:酵母属(Saccharomyces),曲霉属(Aspergillus),木霉属(Trichoderma),和链孢霉属(Neurospora);植物细胞,包括来自以下植物的那些:玉米(com),高粱,烟草,低芥酸油菜,大豆,棉花,番茄,马铃薯,苜蓿,向日葵等;和动物细胞,包括鱼类、鸟类和哺乳动物。适宜的鱼类细胞包括但不限于来自以下物种的那些:鲑鱼,鳟鱼,罗非鱼,金枪鱼,鲤鱼,比目鱼,大比目鱼,旗鱼,鳕鱼和斑马鱼。适宜的鸟类细胞包括但不限于以下动物的细胞:鸡,鸭,鹌鹑,雉和火鸡,和其它丛林竞技或游戏鸟类。适宜的哺乳动物细胞包括但不限于来自以下动物的细胞:马,奶牛,水牛,鹿,绵羊,兔子,啮齿动物如小鼠、大鼠、仓鼠和豚鼠,山羊,猪,灵长目动物,海洋哺乳动物,包括海豚和鲸鱼,以及细胞系,例如任何组织或干细胞类型的人细胞系,和干细胞,包括全能和非全能细胞,和非人类受精卵。
适宜的细胞也包括涉及在众多疾病状态(即使还处于非疾病状态)中的那些细胞类型。因此,适宜的真核细胞类型包括但不限于,所有类型的肿瘤细胞(例如,黑色素瘤,骨髓性白血病,肺癌,乳腺癌,卵巢癌,结肠癌,肾癌,前列腺癌,胰腺癌和睾丸癌),心肌细胞,树突状细胞,内皮细胞,上皮细胞,淋巴细胞(T细胞和B细胞),肥大细胞,嗜酸性粒细胞,血管内膜细胞,巨噬细胞,自然杀伤细胞,红血球,肝细胞,白细胞(包括单核白细胞),干细胞例如造血干细胞、神经肝细胞、皮肤肝细胞、肺肝细胞、肾肝细胞、肝肝细胞和心肌干细胞(用于筛选分化因子和去分化因子),破骨细胞,软骨细胞和其它结缔组织细胞,角化细胞,黑色素细胞,肝细胞,肾细胞,和脂肪细胞。在某些实施方式中,细胞是是原发疾病状态细胞,例如原发肿瘤细胞。适宜的细胞也包括已知的研究细胞,包括但不限于,Jurkat T细胞,NIH3T3细胞,CHO,COS等。参见ATCC细胞系目录,其明确并入本申请以作参考。
在一些实施方式中,靶细胞是肿瘤微囊泡或肿瘤大多孔室脂质体。肿瘤细胞微泡也称为肿瘤分泌的微囊泡或肿瘤分泌的外泌体,其可见于循环血液中并且具有免疫抑制活性。肿瘤细胞微泡的直径尺寸通常为30-200nm。较大的肿瘤微囊泡可以称为肿瘤大囊泡,且直径尺寸可以为3-10μm。
本申请所述的水凝胶颗粒可以用于本领域技术人员已知的任何流式细胞仪。例如,以下表9中给出的一个或多个流式细胞仪可用于本申请所述的水凝胶和试验。
实施例
通过参照以下实施例来进一步说明本发明。但是,应该注意的是,这些实施例与上述实施方式一样是说明性的,并不应以任何方式解释为对本发明的范围进行限制。
实施例1:产生水凝胶颗粒
紫外光刻法的光掩模得自CADart Services Inc.且使用AutoCad(AutoDesk,Inc.)设计。使用校准紫外光源(OAI,Inc.)将SU-8光刻抗蚀剂(Microchem,Inc.)在4″硅片上光交联,以产生微流体装置制造的掩模。PDMS(聚二甲基硅氧烷,Sigma Aldrich,Inc.)使用软光刻法和微流体装置制造的标准公开方法(参见,McDonald JC,et al.,2000,Electrophoresis 21:27-40)制备和成型。
液滴使用流动聚焦几何形成,其中两个油通道汇聚单体水性溶液的中心流,从而打破油包水乳液中的液滴。对于液滴形成,氟碳化合物油(Novec 7500 3M,Inc.)用作外部的连续相液体。为使液滴在聚合前稳定,将表面活性剂以0.5%w/w添加到油相(Krytox 157FSH的羧酸铵盐,Dupont)。为制备碱性聚丙烯酰胺凝胶颗粒,使用单体水性溶液的中心相,该水溶液包含N-丙烯酰胺(1-20%w/v)、交联剂(N,N′-双丙烯酰胺,0.05-1%w/v)、促进剂、和过硫酸铵(1%w/v)。将促进剂(N,N,N′,N′-四甲基乙二胺(2%vol%)添加到油相中,以便于在液滴形成之后触发水凝胶颗粒聚合。
将几种共聚单体添加到碱性凝胶制剂中以增加官能度。在凝胶形成之后,烯丙基胺为二次标记提供伯胺基团。我们通过添加共聚单体丙烯酸烯丙基酯和甲基丙烯酸烯丙基酯来调节凝胶的折射率,从而调节前向散射。液滴的侧向散射如下调节:在聚合之前将二氧化硅纳米粒子和/或PMMA(聚(甲基丙烯酸甲酯))颗粒(~100nm)的胶状悬浮体添加到中心水相。
水凝胶颗粒的化学计量倍增通过利用共聚单体实现,所述共聚单体包含用于二次标记的化学正交侧基(胺,羧基,马来酰亚胺,环氧化物,炔烃等)。
以5kHz的平均速率形成液滴,将其在氟碳化合物油相中收集。聚合在50℃进行30分钟,将所得水凝胶颗粒从油相洗涤到水性溶液中。
实施例2:12 11m水凝胶颗粒的产生和显像
使包含5%丙烯酰胺、0.25%双丙烯酰胺、0.05%烯丙基胺、和0.1%过硫酸铵的水流过中心通道,并由包含0.1%TEMED的油汇聚穿过10微米喷嘴,以制得10μm水凝胶颗粒,如图3A所示。聚合之后,将颗粒在水中洗涤,如图3B所示,并将其缀合于相关的染料。荧光水凝胶颗粒用荧光显微镜显像,如图3C所示。
实施例3:水凝胶颗粒光学性质的多维调节
如图4所述,在多个维度上调节水凝胶颗粒,以匹配不同于聚苯乙烯小珠的具体细胞类型。使用光学参数例如FSC和SSC(图4A)或二次标记物的组合将细胞去卷积。调节水凝胶颗粒以匹配不同于聚苯乙烯小珠(棕色)的具体细胞类型SSC和FSC,其在尺寸(FSC)和侧向散射上是有限度的(图4B)。水凝胶颗粒用化学计量比调节的具体化学侧基和二次标记物进一步官能化,从而使细胞类型精确匹配,而又不会损害生物噪声,依然与固定细胞系行为一样(图4C)。
实施例4:流式细胞仪延迟时间与水凝胶颗粒直径的关系
如图5所示,流式细胞仪的液滴间延迟可与水凝胶颗粒直径精确相关。所示数据为当使用流式细胞仪喷嘴尺寸为70至100μm时,直径为3μm、6μm、10μm、32μm、和50μm的水凝胶颗粒。
实施例5:含有包封的DNA的水凝胶颗粒与细胞的比较
为形成含有包封的DNA的水凝胶颗粒,将40μg/mL-1000μg/mL的重组小牛胸腺DNA添加到包含20%在水中的19∶1(丙烯酰胺:双丙烯酰胺)和0.1%烯丙基胺的聚合物混合物中。在液滴形成前,将0.4%过硫酸铵添加到混合物中。水凝胶颗粒按照实施例1所述形成。当使用商业成像细胞仪和使用碘化丙锭显像时,含有200μg/mL包封的小牛胸腺DNA的水凝胶颗粒显示出细胞样的染色,并将其与使用相同过程染色的中国仓鼠卵巢细胞进行比较。图像使用Nexcelom CellometerTM获得(图6)。
将得自颊粘膜拭子的细胞在PBS中洗涤并用碘化丙锭染色。平行地,包含一系列DNA浓度的水凝胶颗粒的各群落也以相同方式染色。细胞和颗粒悬浮液都在流式细胞仪(488/590nm激发/发射)上分析。面颊细胞和相同量程的包封的DNA颗粒的流式细胞计数法分析表明,颗粒显示出一系列细胞样的荧光性质(图7,左图)。染色的强度显示与中位强度线性相关,通过流式细胞计数法测得(图7,右图)。
实施例6:调节水凝胶颗粒侧向散射
将硅胶以12.5%、6.25%、3.125%和0%添加到聚合物混合物的水性部分,水凝胶颗粒按照实施例1所述形成。前向散射和侧向散射数据使用流式细胞仪获得。结果表明,侧向散射信号(图8,左图)随着包封的纳米粒子的百分增高会增加,而前向散射(图8,右图)总体保持未变,表明与侧向散射和前向散射的调节无关。
实施例7:调节水凝胶颗粒前向散射
在该实验中,在10至40%的范围内改变丙烯酰胺:双丙烯酰胺在水凝胶组合物中的百分比,以调节水凝胶颗粒的折射率,通过在流式细胞仪中的前向散射测得。如图9所示,前向散射随着丙烯酰胺:双丙烯酰胺相对于的百分比增加而增加。
实施例8:调节水凝胶颗粒光学性质
调节水凝胶颗粒以匹配所需细胞亚型的光学性质的实例。可以将共聚单体与纳米粒子组合以在流式细胞仪中使用被动光学测量调节水凝胶的前向散射和侧向散射两种性质。通过将这些性质与化学不稳定的共聚单体(例如烯丙基胺,丙烯酸)组合,可以增加和标记(如果需要)另外的荧光团/蛋白质/生物侧基,以便于除了散射性质之外还匹配细胞子群落染色。这些是三种主要的可利用流式细胞计数法确定细胞的度量。另外的侧基例如包含重金属的那些可以用于例如Cy-TOF(细胞计数法,飞行时间质谱)标定。最后,可以包封生物相容材料以模拟亚细胞细胞器染色。
实施例9:调节水凝胶颗粒光学性质
将50nm纳米粒子胶状悬浮体结合到水凝胶基质中以模拟淋巴细胞和单核细胞的光学性质(图13A和13B)。改变悬浮体的百分比组成以匹配来自血液样本对照的血细胞子群落(Streck)(图13C)。
特别地,调节水凝胶颗粒的丙烯酰胺单体浓度(0.7-0.8M)以增加颗粒的前向散射,从而匹配血细胞子群落。也可以改变双丙烯酰胺交联剂的百分比来影响前向散射(1-5%)。二氧化硅纳米粒子以5%或10%用于组合物来调节侧向散射。该实验的结果显示于图13。
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本申请通篇引用的所有文件、专利、专利申请、出版物、产品说明书、和规程完全并入本申请以作参考,用于所有目的。
本说明书中例证和讨论的实施方式仅意在以发明人所知的最好方式教导本领域技术人员制备和使用本发明。在不背离本发明的情况下,上述本发明实施方式的修改和变型也是可行的,本领域技术人员根据以上教导是可以知晓的。因此应该理解的是,在权利要求及其等价物的范围内,本发明可以按没有具体说明的方式实践。
Claims (50)
1.一种组合物,其包含:
水凝胶颗粒;
水性溶液;
所述水凝胶颗粒悬浮在所述水性溶液中,所述水凝胶颗粒包含聚合的单体并且具有官能化的表面和调节散射的添加剂中的至少一者,使得所述水凝胶颗粒具有基本上相似于靶细胞的对应光学散射性质的光学散射性质,
所述光学散射性质由以下提供:共聚单体、化学侧基、包封材料、胶态二氧化硅或丙烯酰胺与双丙烯酰胺的比率,其中基本上相似表示至少40%相似。
2.权利要求1所述的组合物,其中所述基本上相似于靶细胞的对应光学散射性质的光学散射性质是侧向散射。
3.权利要求1所述的组合物,其中所述基本上相似于靶细胞的对应光学散射性质的光学散射性质是前向散射。
4.权利要求1所述的组合物,其中所述调节散射的添加剂包括共聚单体。
5.权利要求1所述的组合物,其中所述调节散射的添加剂包括纳米粒子的胶态悬浮体。
6.权利要求1所述的组合物,其中所述靶细胞是淋巴细胞、单核细胞、或粒细胞之一。
7.权利要求1所述的组合物,其中所述聚合的单体是可生物降解的单体。
8.权利要求7所述的组合物,其中所述可生物降解的单体是单糖,二糖,多糖,肽,蛋白质,或蛋白质结构域。
9.权利要求7所述的组合物,其中所述可生物降解的单体是结构多糖。
10.权利要求7所述的组合物,其中所述可生物降解的单体是以下之一:琼脂,琼脂糖,海藻酸,微藻多糖酸,α-葡聚糖,支链淀粉,直链淀粉,阿拉伯木聚糖,β-葡聚糖,胼胝质,卡苏兰,角叉菜胶多糖,动物纤维素,纤维素,壳质,壳聚糖,金藻昆布多糖,凝胶多糖,糊精,右旋糖苷,聚蔗糖,果聚糖,岩藻依聚糖,半乳葡甘露聚糖,半乳甘露聚糖,半乳糖胺半乳糖,结冷胶,葡聚糖,葡甘露聚糖,葡萄醛酸木聚糖,糖萼,糖原,半纤维素,同多糖,羟丙甲纤维素,菊粉,开菲尔多糖,海带多糖,蘑菇多糖,果聚糖多糖,地衣多糖,甘露聚糖,混联葡聚糖,裸藻淀粉,果胶酸,果胶,喷他淀粉,植物糖原,平菇葡聚糖,聚葡萄糖,多糖肽,金属卟啉,普鲁兰多糖,裂褶菌多醣,左旋糖,西佐糖,文莱胶,黄原胶,木聚糖,木葡聚糖,酵母聚糖,或其组合。
11.权利要求7所述的组合物,其中所述可生物降解的单体选自纤维糊精和环糊精。
12.权利要求7所述的组合物,其中所述可生物降解的单体是α-环糊精。
13.权利要求7所述的组合物,其中所述可生物降解的单体是艾考糊精。
14.一种组合物,其包含:
悬浮在水性溶液中的水凝胶颗粒,所述水凝胶颗粒包含聚合的单体并且具有衍生化的表面,使得所述水凝胶颗粒具有模拟靶细胞的细胞内组分中的一者或整个靶细胞的对应光学性质的光学性质,所述光学性质由以下提供:共聚单体、化学侧基、包封材料、胶态二氧化硅或丙烯酰胺与双丙烯酰胺的比率。
15.权利要求14所述的组合物,其中所述靶细胞是白细胞。
16.权利要求14所述的组合物,其中所述靶细胞是原核细胞或真核细胞之一。
17.权利要求14所述的组合物,其中所述聚合的单体是可生物降解的单体。
18.权利要求17所述的组合物,其中所述可生物降解的单体是单糖,二糖,多糖,肽,蛋白质,或蛋白质结构域。
19.权利要求14所述的组合物,其中所述水凝胶颗粒的结构性质是二级标记物波形。
20.权利要求19所述的组合物,其中所述二级标记物波形是荧光标记物波形。
21.权利要求14所述的组合物,其中所述聚合的单体是双官能单体。
22.确定样本是否包括淋巴细胞和单核细胞中的一种或多种的方法,其包括:
向细胞计数装置中插入悬浮在水性溶液中的水凝胶颗粒的群体,所述水凝胶颗粒的群体包括:(1)第一部分的水凝胶颗粒,其具有基本上相似于淋巴细胞的前向散射和侧向散射,和(2)第二部分的水凝胶颗粒,其具有基本上相似于单核细胞的前向散射和侧向散射,其中所述水凝胶颗粒的群体的每个水凝胶颗粒包含聚合的单体并且用至少一种光学添加剂官能化;
使用所述细胞计数装置测量所述水凝胶颗粒的群体的每个水凝胶颗粒的前向散射和侧向散射;
基于所述水凝胶颗粒的群体的测量的前向散射和侧向散射标定所述细胞计数装置;
将包含细胞群体的样本添加到所标定的细胞计数装置;和
使用所标定的细胞计数装置测量所述细胞群体的每个细胞的前向散射和侧向散射,以确定所述样本是否包括淋巴细胞和单核细胞中的一种或多种,
其中基本上相似表示至少40%相似。
23.检测样本中的白细胞的方法,其包括:
向细胞计数装置中插入水性溶液中的水凝胶颗粒,所述水凝胶颗粒的光学散射性质基本上相似于白细胞,所述水凝胶颗粒包含聚合的单体并且用至少一种光学添加剂官能化;
使用所述细胞计数装置测量所述水凝胶颗粒的光学散射性质以标定所述细胞计数装置;
将样本插入所述细胞计数装置中,所述样本包含细胞群体;
测量所述细胞群体中的个体细胞的光学散射性质;和
基于光学散射性质的测量结果确定白细胞或其群体是否存在于所述样本中,
其中基本上相似表示至少40%相似。
24.权利要求23所述的方法,其进一步包括:如果确定一个或多个白细胞存在于所述样本中,则将所述白细胞或白细胞群体分选到单独的容器中。
25.权利要求23所述的方法,其进一步包括:向所述细胞计数装置中插入水凝胶颗粒的群体,其中所述水凝胶颗粒的群体的每个水凝胶颗粒具有基本上相似于白细胞亚型的至少一种光学散射性质,其中所述细胞计数装置基于所述水凝胶颗粒的群体的每个水凝胶颗粒的测量的光学散射性质标定。
26.权利要求25所述的方法,其中对于所述水凝胶颗粒的群体中的每一个水凝胶颗粒而言,所述至少一种光学散射性质是相同的。
27.权利要求23所述的方法,其中所述至少一种光学散射性质是侧向散射。
28.权利要求23所述的方法,其中所述至少一种光学散射性质是前向散射。
29.权利要求23所述的方法,其中所述水凝胶颗粒具有基本上相似于白细胞的荧光发射,所述方法进一步包括:
使用所述细胞计数装置测量所述水凝胶颗粒的荧光发射以标定所述细胞计数装置;和
测量所述细胞群体中的个体细胞的荧光发射,其中进一步基于测量的荧光发射确定白细胞或其群体是否存在于所述样本中。
30.权利要求23所述的方法,其中所述至少一种光学散射性质是FSC与SSC的比率。
31.权利要求23所述的方法,其中所述水凝胶颗粒的聚合的单体选自:甲基丙烯酸羟基乙基酯,甲基丙烯酸乙基酯,甲基丙烯酸2-羟基乙基酯,甲基丙烯酸丙二醇酯,丙烯酰胺,N-乙烯基吡咯烷酮,甲基丙烯酸甲基酯,甲基丙烯酸缩水甘油基酯,甲基丙烯酸甘油酯,甲基丙烯酸乙二醇酯,乙二醇,富马酸,甲基丙烯酸2-羟基乙基酯,甲基丙烯酸羟基乙氧基乙基酯,甲基丙烯酸羟基二乙氧基乙基酯,甲基丙烯酸甲氧基乙基酯,甲基丙烯酸甲氧基乙氧基乙基酯,甲基丙烯酸甲氧基二乙氧基乙基酯,聚(乙二醇)甲基丙烯酸酯,甲氧基-聚(乙二醇)甲基丙烯酸酯,甲基丙烯酸,甲基丙烯酸钠,甲基丙烯酸甘油酯,甲基丙烯酸羟基丙基酯,甲基丙烯酸羟基丁基酯,丙烯酸苯基酯,甲基丙烯酸苯基酯,丙烯酸苄基酯,甲基丙烯酸苄基酯,丙烯酸2-苯基乙基酯,甲基丙烯酸2-苯基乙基酯,丙烯酸2-苯氧基乙基酯,甲基丙烯酸2-苯氧基乙基酯,丙烯酸苯基硫代乙基酯,甲基丙烯酸苯基硫代乙基酯,丙烯酸2,4,6-三溴苯基酯,甲基丙烯酸2,4,6-三溴苯基酯,丙烯酸五溴苯基酯,甲基丙烯酸五溴苯基酯,丙烯酸五氯苯基酯,甲基丙烯酸五氯苯基酯,丙烯酸2,3-二溴丙基酯,甲基丙烯酸2,3-二溴丙基酯,丙烯酸2-萘基酯,甲基丙烯酸2-萘基酯,丙烯酸4-甲氧基苄基酯,甲基丙烯酸4-甲氧基苄基酯,丙烯酸2-苄氧基乙基酯,甲基丙烯酸2-苄氧基乙基酯,丙烯酸4-氯苯氧基乙基酯,甲基丙烯酸4-氯苯氧基乙基酯,丙烯酸2-苯氧基乙氧基乙基酯,甲基丙烯酸2-苯氧基乙氧基乙基酯,N-苯基丙烯酰胺,N-苯基甲基丙烯酰胺,N-苄基丙烯酰胺,N-苄基甲基丙烯酰胺,N,N-二苄基丙烯酰胺,N,N-二苄基甲基丙烯酰胺,N-二苯基甲基丙烯酰胺,N-(4-甲基苯基)甲基丙烯酰胺,N-1-萘基丙烯酰胺,N-4-硝基苯基丙烯酰胺,N-(2-苯基乙基)丙烯酰胺,N-三苯基甲基丙烯酰胺,N-(4-羟基苯基)丙烯酰胺,N,N-甲基苯基丙烯酰胺,N,N-苯基苯基乙基丙烯酰胺,N-二苯基甲基甲基丙烯酰胺,N-(4-甲基苯基)甲基甲基丙烯酰胺,N-1-萘基甲基丙烯酰胺,N-4-硝基苯基甲基丙烯酰胺,N-(2-苯基乙基)甲基丙烯酰胺,N-三苯基甲基甲基丙烯酰胺,N-(4-羟基苯基)甲基丙烯酰胺,N,N-甲基苯基甲基丙烯酰胺,N,N'-苯基苯基乙基甲基丙烯酰胺,N-乙烯基咔唑,4-乙烯基吡啶,2-乙烯基吡啶,或其组合。
32.权利要求23所述的方法,其中所述水凝胶颗粒的聚合的单体是可生物降解的单体。
33.权利要求32所述的方法,其中所述可生物降解的单体是单糖,二糖,多糖,肽,蛋白质,或蛋白质结构域。
34.权利要求33所述的方法,其中所述可生物降解的单体是包含至少一个非天然氨基酸的蛋白质或蛋白质结构域。
35.权利要求32所述的方法,其中所述可生物降解的单体是结构多糖。
36.权利要求32所述的方法,其中所述可生物降解的单体是琼脂,琼脂糖,海藻酸,微藻多糖酸,α-葡聚糖,支链淀粉,直链淀粉,阿拉伯木聚糖,β-葡聚糖,胼胝质,卡苏兰,角叉菜胶多糖,动物纤维素,纤维素,壳质,壳聚糖,金藻昆布多糖,凝胶多糖,糊精,右旋糖苷,聚蔗糖,果聚糖,岩藻依聚糖,半乳葡甘露聚糖,半乳甘露聚糖,半乳糖胺半乳糖,结冷胶,葡聚糖,葡甘露聚糖,葡萄醛酸木聚糖,糖萼,糖原,半纤维素,同多糖,羟丙甲纤维素,菊粉,开菲尔多糖,海带多糖,蘑菇多糖,果聚糖多糖,地衣多糖,甘露聚糖,混联葡聚糖,裸藻淀粉,果胶酸,果胶,喷他淀粉,植物糖原,平菇葡聚糖,聚葡萄糖,多糖肽,金属卟啉,普鲁兰多糖,裂褶菌多醣,左旋糖,西佐糖,文莱胶,黄原胶,木聚糖,木葡聚糖,酵母聚糖,或其组合。
37.权利要求32所述的方法,其中所述可生物降解的单体选自纤维糊精和环糊精。
38.权利要求32所述的方法,其中所述可生物降解的单体是α-环糊精。
39.权利要求32所述的方法,其中所述可生物降解的单体是艾考糊精。
40.权利要求32所述的方法,其中所述可生物降解的单体是壳聚糖或透明质酸。
41.权利要求33所述的方法,其中所述蛋白质是结构蛋白,其结构域,或其组合。
42.权利要求33所述的方法,其中所述蛋白质是蛋白多糖,其结构域,或其组合。
43.权利要求33所述的方法,其中所述蛋白质是细胞外基质组分。
44.权利要求42所述的方法,其中所述蛋白多糖是核心蛋白多糖,双糖链蛋白多糖,睾丸蛋白聚糖,双库尼茨抑制剂,纤维调节素,光蛋白聚糖,其结构域,或其组合。
45.权利要求43所述的方法,其中所述蛋白质是弹性蛋白或蛋白多糖。
46.权利要求43所述的方法,其中所述蛋白质是胶原蛋白。
47.权利要求46所述的方法,其中所述胶原蛋白是胶原蛋白I型,胶原蛋白II型,胶原蛋白III型,其结构域或其组合。
48.权利要求23所述的方法,其中所述水凝胶颗粒的聚合的单体是双官能的。
49.权利要求23所述的方法,其中所述至少一种光学添加剂是表4中列出的细胞表面标记物之一,或其组合。
50.标定用于分析白细胞的细胞计数装置的方法,其包括:
向细胞计数装置中插入悬浮在水性溶液中的水凝胶颗粒的群体,所述水凝胶颗粒的群体的每个水凝胶颗粒具有基本上相似于淋巴细胞、单核细胞、或粒细胞之一的侧向散射和前向散射,每个水凝胶颗粒包含聚合的单体并且用至少一种光学添加剂官能化;
使用所述细胞计数装置测量所述水凝胶颗粒的群体的每个水凝胶颗粒的侧向散射和前向散射;和
基于所述水凝胶颗粒的群体的测量的侧向散射和前向散射标定用于分析白细胞的细胞计数装置,
其中基本上相似表示至少40%相似。
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