CN114486830A - 用于单细胞中单分子蛋白质与生物分子计数的系统与方法 - Google Patents
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Abstract
本发明属于生命科学技术领域,具体为用于单细胞中单分子蛋白质与生物分子计数的系统与方法。本发明系统包括微注射泵、三维可移动定位样品操作平台、荧光显微镜物镜系统、CCD图像采集系统、荧光图像处理和计数系统、抗体功能化修饰的玻片;在显微镜监视下使用样品操作平台控制吸取皮升体积单细胞,加样到功能化玻片上;在样品微液滴原位进行细胞裂解及目标分子释放,并被玻片上抗体捕集形成单分子层;标记荧光标签后,由荧光成像系统拍摄并完成荧光计数。本发明单次成像可实现对单细胞内1到3000个分子的蛋白质的精准计数,通过多次、多视野拍摄和数据叠加扩大计数范围至10^4。本发明可扩展对皮升级液滴内分子进行分子计数。
Description
技术领域
本发明属于生命科学技术领域,具体涉及用于单细胞中单分子蛋白质与生物分子精确计数的系统与方法。
背景技术
蛋白质作为所有生物的重要组成部分和功能承担着者,参与从DNA复制到基本细胞功能实现等的全部过程。单个细胞中的蛋白质的组成和丰度保证了其独特的细胞功能和行为,特别是一些超低拷贝数目的蛋白质(单个细胞内拷贝数低于1000)在细胞信号网络中发挥起到关键作用,同时也影响着细胞的一些重要的生理学和病理学行为。然而这些单细胞中的蛋白难以使用传统的、基于大量样品的蛋白质定量方法进行定量,这些信息的缺失严重影响我们对一些关键的生命过程的理解,尤其是一些与疾病的发生、发展相关的过程。
目前单细胞蛋白质的定量方法采用的大多是基于常规蛋白质定量方法的改进和创新,具有这些方法的一切限制,最重要的是仍无法对低拷贝蛋白数目蛋白进行精准定量。这些方法甚至需要进行单细胞化的适配,增加了额外的设备和实验流程,花费高,学习曲线长。近年来也有研究者尝试设计新的单细胞蛋白质定量方法解决此问题,如Guan等报道了一种基于激光诱导荧光的低成本平台,用于对细胞内的可溶性蛋白进行定量;A.Salehi-Reyhani等开发了一种通过对荧光图像进行化学计量分析对单细胞细胞内蛋白进行绝对定量的方法。这些方法虽然实现了对单细胞内低拷贝蛋白的定量,但他们都将定量的目标蛋白限制在了细胞内蛋白,这也限制了这些方法的进一步推广。
基于以上认识,为了解决单细胞低拷贝数目蛋白的计数问题,本发明设计了一种用于蛋白质与生物分子计数的系统,以皮升级液体体积对任意靶细胞进行取样,尽可能地降低样品损失并提高了目标蛋白的浓度;同时结合荧光成像和计数方法,完成对单细胞内任意蛋白分子及生物分子进行计数。平台结构简单,操作学习曲线短,利于生命科学中的应用和推广。
发明内容
本发明的目的在于提供一种结构简单、操作方便且利于推广的于单细胞中单分子蛋白质与生物分子计数的系统及方法。
本发明提供的于单细胞中单分子蛋白质与生物分子计数的系统,包括:微注射泵1、三维可移动定位样品操作平台2、摄像头4、荧光显微镜物镜系统5、CCD图像采集系统6、荧光图像处理和计数系统7、玻片8;其中:
所述微注射泵1 连接有微量注射器,微注射泵1用于精准控制皮升级体积的单细胞或液体吸取和排出;
所述三维可移动定位样品操作平台2,设置有带尖端的毛细管3,毛细管3 的内径为10-50微米,用于吸取含有单个细胞的皮升级液体样品;该样品操作平台2可作三维移动,并对样品进行精确定位;其中,毛细管3与微注射泵1连接;
所述荧光显微镜物镜系统5,用于实时观察样品操作过程及毛细管内液体液面的变化;在荧光成像阶段用于目标分子单分子层的定位和辅助成像;
所述玻片8,经过抗体功能化修饰,其表面覆盖有非水溶性油层(或油滴);含有单细胞的微液滴样品放置于油层之下;所述玻片8放置于荧光显微镜物镜系统5的物镜台上;
所述CCD图像采集系统6,其功能为实现目标生物分子的荧光成像;
所述荧光图像处理和计数系统7,用于对所拍摄的荧光图像进行处理和荧光分子计数;
所述辅助摄像头4,设置于所述玻片8的上方且有一定的倾斜角度,用于辅助观察实时观察样品操作过程及毛细管内液体液面的变化;
所述荧光图像处理和计数系统7依次与所述CCD图像采集系统6、荧光显微镜物镜系统5的物镜台连接;
各部件通过三维移动架设在荧光显微镜系统上。
本发明设计的用于单细胞中单分子蛋白质与生物分子计数的系统,实现单细胞蛋白质与生物分子计数的原理如下:在显微镜和辅助摄像头实时观察下,由三维样品操作平台2操控,由微注射泵1和毛细管3配合,从细胞池吸取目标单细胞,目标单细胞的体积可以为5-500皮升(任意体积);随后转移并加样到覆盖有油层(或油滴)的功能化玻片的指定位置,在油层(或油滴)内的含细胞微液滴进行细胞裂解及释放目标分子;目标分子被修饰在玻片上的抗体亲和捕集形成单分子层。随后标记耦合抗体的荧光标签,使用荧光成像系统进行拍摄,并完成目标分子的荧光计数与定量测定。
使用本发明设计的皮升级液体操作系统进行单细胞中单分子蛋白质与生物分子计数的方法,具体步骤如下:
(1)单细胞样品取样:在显微镜直视和辅助摄像头实时观察下,使用三维可移动定位样品操作平台2,控制毛细管3移动,进行精准定位,微注射泵1工作,与之相连毛细管3的尖端从细胞池吸取设定体积(5-500 pL)的含有单个目标细胞的皮升级样品溶液,并将样品溶液转移到表面覆盖油层或油滴的抗体功能化修饰的玻片的指定加样位点,完成加样;
(2)油层(或油滴)内细胞原位裂解:室温静置1-120分钟等待油层内含有单细胞的样品微液滴中的细胞完全裂解;
(3)目标分子固定:将已经完成原位液滴内细胞裂解的、覆盖油层的功能化玻片置于25-37℃环境中反应1-120分钟,以促进目标蛋白分子或其他目标生物分子固定在玻片上并形成单分子层;
(4)荧光标签的标记:原位加入耦合抗体荧光标签,室温下反应1-120分钟;
(5)荧光成像及分子计数:清洗未结合荧光标签后,封片;使用荧光显微镜拍摄荧光照片,计数荧光点数目,完成单细胞内目标分子的分子计数。
本发明中,功能化玻片上加载有不与水滴相容的油滴或油层,使皮升级液体不能在单细胞裂解、目标分子固定和荧光标记过程中挥发、移动。加样过程在油层下进行,直接将皮升级样品液滴加在功能化玻片表面。
本发明中,内单细胞微液滴在油层(或油滴)中单细胞裂解,其中,裂解试剂的加入方式为:在待取样的细胞溶液中预先加入裂解试剂,也可用带尖端毛细管吸取裂解试剂加入单细胞液滴进行原位裂解。
本发明可以对单细胞样品任意蛋白质组分(包括细胞膜和细胞内部)进行计数,单次拍摄成像可以实现对单细胞内1到3000个分子数的蛋白质的精准计数,并可通过多次、多视野拍摄和数据叠加进行动态范围扩展,将计数范围扩大至1-10^4范围。本发明还可以扩展到对皮升级液滴内分子进行分子计数和绝对定量。对于单细胞重要信号网络和通路中的关键节点蛋白的定量有着重要作用;同时可以对细胞低表达的重要蛋白质标志物(特别是肿瘤细胞)进行定量对疾病的诊断、治疗方案的选择、治疗结果和预后的评估意义重大,有着广阔的生物学和临床医学应用前景。例如本发明的用于单细胞蛋白质与生物分子计数的皮升级液体操作平台,曾对肠息肉病人外周血CD8+T细胞单细胞的TCF-1蛋白分子进行了计数,发现单细胞TCF-1分子表达数目跨度巨大(78-1790),细胞间存在显著异质性。本发明对于单细胞蛋白质组学研究的方法学是一个重要补充。
附图说明
图1为用于单细胞中单分子蛋白质与生物分子计数的皮升级液体操作平台及使用方法工作流程示意图。
图2为包含单细胞采样、油滴下单细胞裂解和目标蛋白质分子固定的过程。
图3为100X荧光显微镜拍摄荧光图像(A)与自动识别的荧光标记分子计数编号(B)。
图4为使用本发明对肠息肉患者外周血CD8+T细胞的TCF-1的分子数目的计数。
图中标号:1为微注射泵,2为三维样品操作平台,3为毛细管,4为摄像头,5为显微镜物镜,6为CCD图像采集系统,7为荧光图像处理和计数系统,8为覆盖油层的抗体功能化玻片,9为含有单细胞的微液滴样品,10为细胞,11为固定在抗体功能化玻片上的目标蛋白单分子层。
具体实施方式
通过实施例对本发明提供的用于单细胞中单分子蛋白质与生物分子计数的皮升级液体操作平台及使用方法在肠息肉病人外周血CD8+T细胞TCF-1分子的计数的应用做出进一步说明。
1、用于单细胞中单分子蛋白质与生物分子计数的皮升级液体操作系统的构建
皮升级单细胞毛细管样品操作系统选取30厘米长、40微米内径的毛细管通过无死体积两通管与1微升微量注射器相连,并固定在双向微量注射泵上,设定单次吸样和推样的体积为50皮升,并将系统连同三维平台固定在荧光显微镜上,并设立辅助摄像头实时观察毛细管尖端位置和毛细管内液面变化。将抗体功能化玻片做好标记,并在其表面覆盖全氟油备用。将iXonUltra 897 EMCCD连接于荧光显微镜用于荧光成像拍摄。参见图3所示。
2、单个CD8+T细胞的取样
纯化获得CD8+T细胞后,将细胞溶剂置换为含有细胞裂解液组分的温和的单细胞裂解液,细胞浓度保持在10个/微升,使用皮升级-单细胞毛细管样品操作系统以50皮升固定体积进行单细胞采样,后迅速将细胞转移并滴加在功能化玻片的特定加样位置。
3、原位液滴内细胞裂解
将表面覆盖油层的、已经加样的功能化玻片置于室温静置15分钟等待单细胞样品完全裂解。
4、目标蛋白固定
将已经完成原位液滴内细胞裂解的、覆盖油的功能化玻片置于37℃环境中反应1小时,完成目标蛋白分子在玻片上的固定。
5、荧光标签的标记
原位加入5微升浓度为500 pM耦合抗体的量子点荧光标签,室温下反应1小时。
6、荧光成像及绝对计数
清洗未结合量子点荧光标签后,封片。上机使用荧光显微镜拍摄荧光照片,光电倍增倍数设定为300,曝光时间100毫秒。计数荧光点数目,完成单细胞内目标蛋白的绝对定量,结果参见图4所示。
Claims (3)
1.一种用于单细胞中单分子蛋白质与生物分子计数的系统,其特征在于,包括:微注射泵、三维可移动定位样品操作平台、辅助摄像头、荧光显微镜物镜系统、CCD图像采集系统、荧光图像处理和计数系统、玻片;其中:
所述微注射泵连接有微量注射器,微注射泵用于精准控制皮升级体积的单细胞或液体吸取和排出;
所述三维可移动定位样品操作平台,设置有带尖端的毛细管,毛细管的内径为10-50微米,用于吸取含有单个细胞的皮升级液体样品;该样品操作平台可作三维移动,并对样品池中单个细胞进行精确对准、定位;其中,毛细管与微注射泵连接,可将单个细胞通过毛细管的尖端吸入毛细管内;
所述荧光显微镜物镜系统,用于实时观察样品操作过程及毛细管内液体液面的变化;在荧光成像阶段,用于目标分子单分子层的定位和辅助成像;
所述玻片,经过抗体功能化修饰,在使用时将其表面覆盖上非水溶性油层或油滴;含有单细胞的微液滴样品放置于油层或油滴之下与玻片接触;所述玻片放置于荧光显微镜物镜系统的物镜台上;
所述CCD图像采集系统,用于实现目标生物分子的荧光成像计数;
所述荧光图像处理和计数系统,用于对所拍摄的荧光图像进行处理和荧光分子计数;
所述辅助摄像头,设置于所述玻片的上方且有一定的倾斜角度,用于辅助实时观察样品操作过程及毛细管内液体液面的变化;
所述荧光图像处理和计数系统依次与所述CCD图像采集系统、荧光显微镜物镜系统的物镜台连接;
各部件通过三维移动平台架设在荧光显微镜系统上。
2.一种基于权利要求1所述的系统进行单细胞中单分子蛋白质与生物分子计数的方法,其特征在于,具体步骤如下:
(1)单细胞样品取样:在显微镜和辅助摄像头观察下,使用三维可移动定位样品操作平台,控制毛细管移动,进行精准定位,微注射泵工作,与之相连毛细管的尖端从细胞池吸取设定体积的含有单个目标细胞的皮升级样品溶液,并将样品溶液转移到表面覆盖油层或油滴的抗体功能化修饰的玻片的指定加样位点,完成加样;
(2)油层内细胞原位裂解:室温静置1-120分钟等待油层内含有单细胞的样品微液滴中的细胞完全裂解;
(3)目标分子固定:将已经完成原位液滴内细胞裂解的所述玻片置于25-37℃环境中反应1-120分钟,以促进目标蛋白分子或其他目标生物分子固定在玻片上并形成单分子层;
(4)荧光标签的标记:原位加入耦合抗体荧光标签,室温下反应1-120分钟;
(5)荧光成像及分子计数:清洗未结合荧光标签后,封片;使用荧光显微镜拍摄荧光照片,计数荧光点数目,完成单细胞内目标分子的分子计数。
3. 根据权利要求2所述的方法,其特征在于,所述设定的体积为5-500 pL。
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