CN118169392A - 一种面向高精度分析外泌体表型的超分辨率三色荧光定位方法 - Google Patents
一种面向高精度分析外泌体表型的超分辨率三色荧光定位方法 Download PDFInfo
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Abstract
本发明公开了一种面向高精度分析外泌体表型的超分辨率三色荧光共定位方法,该方法能够去除由于非特异性吸附产生的荧光信号;该方法结合了目标外泌体的三色荧光标记、三色超分辨率成像和像素计数,像素计数方法是通过MATLAB实现的,可以在单像素水平上消除非特异性结合位点;另外,该方法还实现了外泌体膜蛋白的图谱鉴定。通过标记三种特定蛋白质以获得各种外泌体的3D表型信息;本发明实现了根据蛋白质表达水平对不同种类的外泌体进行分类;利用SMLM显微镜和像素计数方法,可以最大限度地提高检测的可靠性。
Description
技术领域
本发明涉及外泌体检测、超分辨成像以及科学计算领域,特别涉及一种面向高精度分析外泌体表型的超分辨率三色荧光定位方法。
背景技术
近年来,外泌体已成为癌症最有前途的生物标志物之一。外泌体是一种细胞外囊泡,直径为30-150nm。它们参与细胞通讯、细胞迁移、血管生成和肿瘤细胞生长的过程,并稳定携带蛋白质、脂质、核酸和其他生物活性物质。外泌体膜蛋白的表达水平在生物学功能中起着重要作用。例如,乳腺肿瘤至少有三种根据肿瘤细胞分子谱分类的亚型。来源于其亲本细胞的外泌体的表面蛋白具有特异性,可以反映亲本细胞的生理信息。到目前为止,各种外泌体蛋白已被用于识别肿瘤起源或亚型,以及区分惰性和高风险病变。因此,基于外泌体特异性蛋白对癌症细胞表型进行分析和分类的技术需求很大。
荧光免疫测定法是外泌体检测的典型方法。荧光免疫标记是表征表面蛋白表达的主要方法。荧光免疫标记基于抗原-抗体反应的原理。荧光抗体则是通过用荧光素标记某些抗原或抗体产生的。然后,将荧光抗体用作探针,与靶向蛋白形成抗原-抗体复合物,以检测组织、细胞或外泌体中的相应抗原(或抗体)。抗体可以在相应激光的激发下产生荧光信号,因此样本可以被可视化。此外,靶向蛋白的定性和定量分析通常通过荧光信号的变化来实现。荧光免疫标记方法由于无放射性、操作简单、灵敏度高和选择性好,可以应用于各种生物分子的检测。免疫测定中的非特异性吸附是不可避免的,并影响测定结果的可靠性。
由于外泌体的体积小和阿贝衍射极限,传统光学显微镜的空间分辨率仅达到200-300nm,不适合观察外泌体。因此,无法通过传统光学显微镜观察外泌物的特异性和非特异性结合点。单分子定位显微镜(SMLM)是一种超分辨率光学技术,分辨率可达20nm。考虑到外泌体的纳米尺寸,使用SMLM进行免疫反应的视觉分析是最合适的。
但是目前的单色荧光定位和双色荧光定位技术仍旧存在假阳性率高的问题,如何去除非特异性吸附导致的假阳性,以及如何提高准确度仍亟待解决。
发明内容
发明目的:本发明为了进一步提高外泌体检测的可靠性,提供了一种超分辨率三色荧光共定位(SR-TFC)方法,并开发了一种基于共定位荧光像素点的外泌体蛋白图谱新策略。本发明不仅提供了金纳米球阵列基底的制备,还提供了三色荧光共定位策略,以及通过特异性计数共定位荧光像素点的方法。
技术方案:为了解决上述技术问题,本发明提供了一种面向高精度分析外泌体表型的超分辨率三色荧光共定位方法,包括如下步骤:
1)在制得的金纳米球阵列基底上修饰捕获探针;
2)在经过步骤1)处理后的金纳米球阵列基底上滴加外泌体溶液进行外泌体捕获;
3)在经过步骤2)处理后的金纳米球阵列基底上滴加膜染料,进行外泌体染色;
4)在经过步骤3)处理后的金纳米球阵列基底上滴加带有荧光的特异性识别探针进行特异性识别;
5)将样品放于单分子定位显微镜下对FAM通道、膜染料通道和Cy5通道同时进行成像,激发光波长分别为488nm、561nm和642nm,同时收集荧光信号并进行合成;
6)应用基于MATLAB的CFPP方法对步骤5)得到的三色超分辨图像进行共定位荧光像素点进行计数,所述CFPP方法是采用MATLAB的运行代码实现,以RGB色彩模式的原理为基础,在MATLAB设置像素点的R、G、B代表着颜色的三个基本分量,三个基本分量的取值范围均为0~255,不同的R、G、B组合会产生不同的颜色,CFPP通过遍历荧光图像中的所有像素点,并将R、G、B三个分量的值均大于等于200的像素点(即255≥R≥200,255≥G≥200,255≥B≥200)记为白色像素点,继而统计出荧光图片中白色像素点的个数。
本发明中像素点200作为一个阈值,可以认为是中等亮度以上,这样可以确保识别出的白色不会包含太多暗色调的像素点,避免计入非特异性免疫结合位点(即由于背景噪声、杂质导致的亮点)。RGB色彩模式中,理论上R=G=B=255时为纯白色。但在实际应用中,由于显示器的校准、环境光线、设备的差异等因素,需要对颜色进行调整以达到视觉上的白色。选择200作为阈值,是在实践中发现的一个合适的平衡点。本发明也通过后续的实验证明选择像素点200将能够更为准确地区分不同的外泌体结合蛋白。
其中,步骤1)中所述金纳米球阵列基底的制备方法如下:通过热蒸发在硅晶片上沉积铬层,通过气液界面加载技术,在所得的镀铬的硅片上紧密排列一层聚苯乙烯微球,通过热蒸发在所得的聚苯乙烯微球阵列的表面沉积一层金层。
其中,所述气液界面加载技术具体步骤为:将聚苯乙烯微球溶液与无水乙醇混合,并超声处理,将混合溶液通过载玻片滑落到去离子水表面,在气液界面形成单层微球膜,滴加十二烷基硫酸钠溶液以固定微球膜,随后,将微球膜从水面转移到硅片上,并在室温下干燥,得到聚苯乙烯微球阵列。
其中,所述铬层的厚度为15nm,所述金层的厚度为15nm。
其中,所述聚苯乙烯微球溶液与无水乙醇以体积比2:1,所述聚苯乙烯微球溶液浓度为5wt%。
其中,步骤1)中所述捕获探针为CD63适配体,其序列为5′-6FAM-CAC CCC ACC TCGCTC CCG TGA CAC TAA TGC TAT TTT TT-(CH2)6-SH-3′。
其中,步骤2)中外泌体溶液包括MDA-MB-231、SKBR3和MCF7外泌体溶液中的一种或几种。
其中,步骤2)中MDA-MB-231外泌体溶液的浓度为7.124×104~7.124×107个/mL,所述SKBR3外泌体溶液的浓度为2.129×104~2.129×107个/mL,所述MCF7外泌体溶液浓度为1.014×104~1.014×107个/mL。
其中,步骤4)中所述特异性识别探针包括Cy5-PD-L1适配体、Cy5-HER2适配体和Cy5-EpCAM适配体,
Cy5-PD-L1适配体序列为:5′Cy5-TAC AGG TTC TGG GGG GTG GGT GGG GAA CCTGTT-3′;
Cy5-HER2适配体序列为:5′Cy5-TTG GGC CGT CGA ACA CGA GCA TGG TGC GTGGAC CTAGGA TGA CCT GAG TAC TGT CCT-3′;
Cy5-EpCAM适配体序列为:5′Cy5-TTC ACT ACA GAG GTT GCG TCT GTC CCA CGTTGT CAT GGG GGG TTG GCC TGT-3′。
其中,所述捕获探针、外泌体、膜染料、特异性识别探针均溶解于PBS缓冲溶液中,所述PBS缓冲液pH=7.2-7.4,浓度为10mM。
本发明提出的超分辨率三色荧光共定位(SR-TFC)方法,可用于外泌体检测和定位。本发明的基底不是使用电子束光刻(EBL)来制备,而是通过自组装方法,简化了检测基底的制备过程。SR-TFC利用经典的三明治型免疫测定方案,指的是通过SMLM对捕获探针、外泌体和免疫探针进行共定位。外泌体、捕获探针和识别探针分别用三种不同的荧光染料标记。对于特异性免疫结合位点,外泌体、捕获探针和识别探针的空间位置高度重叠,而对于非特异性结合位点,外泌体、捕捉探针和识别探针的位置不重叠。
有益效果:与现有技术相比,本发明具备以下优点:本发明将超分辨率三色荧光共定位和像素计数方法相结合,对肿瘤外泌体进行三次标记,并在一个像素中同时检测三个荧光信号,然后使用基于MATLAB的像素计数方法,最大限度地提高检测的可靠性。此外,还实现了外泌体膜蛋白的图谱的鉴定。这种策略可以在单像素水平上消除非特异性结合位点。为了实现多重表型分析,标记程序化细胞死亡配体1(PD-L1)适体、上皮细胞粘附分子(EpCAM)适体和人表皮生长因子受体2(HER2)三种特异性蛋白质以获得各种外泌体的3D表型信息,并对不同种类的外泌体进行分类。
附图说明
图1为本发明的SR-TFC方法和原理示意图。
图2为金纳米球阵列基底的制备过程示意图;
图3为金纳米球阵列基底的电镜图;
图4为不同浓度的MDA-MB-231、SKBR3、MCF 7外泌体和对照组的共定位荧光像素点数拟合曲线图;
图5PD-L1、EpCAM和HER2蛋白在不同种类的外泌体(MDA-MB-231、SKBR3、MCF7)上的表达水平热图;
图6三色荧光共定位策略和双色荧光共定位策略的可靠性。(a)捕获探针和识别探针非特异性吸附导致的假阳性像素点(b)阳性像素点和外泌体或识别探针非特异性吸附导致的假阳性像素点。(c)三色荧光共定位策略、双色共定位策略以及常规单色荧光检测的CFPP结果(直方图)。双色共定位策略和常规单色荧光检测的假阳性率(折线图)。
具体实施方式
本发明实施例中的PBS缓冲液为pH=7.2-7.4,浓度为10mM的PBS缓冲液;FAM-CD63适配体-SH、Cy5-PD-L1适配体、Cy5-HER2适配体和Cy5-EpCAM适配体均由Sangon Biotech公司合成,序列如下;其余材料均为市售所得。
捕获探针:
FAM-CD63-SH;5′-6FAM-CAC CCC ACC TCG CTC CCG TGA CAC TAA TGC TAT TTTTT-(CH2)6-SH-3′;
识别探针1:Cy5-PD-L1:5′-Cy5-TAC AGG TTC TGG GGG GTG GGT GGG GAA CCTGTT-3′;
识别探针2:Cy5-HER2:5′-Cy5-TTG GGC CGT CGA ACA CGA GCA TGG TGC GTG GACCTA GGA TGA CCT GAG TAC TGT CCT-3′
识别探针3:Cy5-EpCAM:5′-Cy5-TTC ACT ACA GAG GTT GCG TCT GTC CCA CGTTGT CAT GGG GGG TTG GCC TGT-3′
实施例1:金纳米球阵列基底的制备
本实施例以有序的聚苯乙烯微球阵列为模板,经氧等离子体刻蚀和热蒸发涂层制备了基底。
具体步骤如下:单晶硅片首先通过标准RCA清洁步骤进行清洁。然后,通过热蒸镀在硅片上沉积一层厚度为15nm的铬。将5wt%的聚苯乙烯微球溶液与无水乙醇以体积比2:1的比例混合,并置于超声波清洗机中处理10分钟得到混合溶液。将混合溶液通过载玻片滑落到去离子水表面,在气液界面形成单层微球膜。在单层微球膜滴加20μL十二烷基硫酸钠(SDS)溶液(2.5wt%)以固定微球膜。随后,将微球膜从水面转移到硅片上,并在室温下干燥,得到聚苯乙烯微球阵列。干燥后,通过使用箱式真空镀膜机在聚苯乙烯微球阵列的表面沉积一层厚度为15nm的金。最后,在1000℃下退火2小时,即可得金纳米球阵列基底。
图2为金纳米球阵列基底的制备过程示意图;图3为金纳米球阵列基底的电镜图。实施例2:外泌体的定量分析和可靠性分析
取5μL捕获探针溶液(FAM-CD63-SH,10μM)滴在实施例1制备的金纳米球阵列基底上,并置于4℃的湿盒中过夜反应。并重复一次该步骤。接着取5μL 1%BSA滴在基底上静置1小时以封闭基底上的多余位点。分别取5μL不同浓度的外泌体溶液(包括浓度分别为7.124×107个/mL、7.124×106个/mL、7.124×105个/mL、7.124×104个/mL的MDA-MB-231外泌体溶液;2.129×107个/mL、2.129×106个/mL、2.129×105个/mL、2.129×104个/mL的SKBR3外泌体溶液;1.014×107个/mL、1.014×106个/mL、1.014×105个/mL、1.014×104个/mL的MCF7外泌体溶液)滴在基底上,并在25℃下反应60分钟。随后,取5μL DiI膜染料溶液滴在基底上,在25℃下反应15分钟。最后,分别取5μL识别探针1Cy5-PD-L1、识别探针2Cy5-HER2或识别探针3Cy5-EpCAM(10μM)滴在基底上,并在置于25℃环境中反应90分钟。在此过程中,识别探针、BSA、外泌体和DiI膜染色剂全部溶解在PBS缓冲液中。捕获探针为CD63适配体,识别探针分别为PD-L1、HER2或EpCAM适配体。每个步骤完成后,均用PBS冲洗。
由于金纳米球阵列,荧光信号表现为阵列点。随着外泌体浓度的增加,DiI和Cy5的信号也增加,因为可以捕获更多的外泌体和识别探针。对于更高的外泌体浓度(107个/mL),可以观察到更多的白色像素。随着外泌体浓度的降低,白色像素的数量逐渐减少。特别是,当外泌体溶液浓度为104个/mL时,在合并的图像中只有几个白色像素。这是由于存在的外泌体较少,形成的三明治型免疫复合物结构较少。
为了更好地量化这些外泌体,用CFPP方法对不同浓度外泌体的合并SMLM图像进行分析。所述的CFPP为通过在Matlab(R2023a)中编写一段代码,对FAM、DiI和Cy5通道的所有合并图像中的共定位荧光像素进行计数。其中,CFPP运行代码的编写是以RGB色彩模式的原理为基础,在计算机图形学中,像素点的Red(R)、Green(G)、Blue(B)代表着颜色的三个基本分量。三个分量的取值范围都是是0(无)到255(最大亮度),不同的R、G、B组合会产生不同的颜色。CFPP通过遍历荧光图像中的所有像素点,并将R、G、B三个分量的值均大于等于200的像素点记为白色像素点,继而统计出荧光图片中白色像素点(即特异性免疫结合位点)的个数。结果如图4所示。图4显示了CFPP计数的SR-TFC像素数,其与外泌体浓度呈正相关。通过拟合曲线,计算出每种类型的外泌体的检测限分别为7.124×104个颗粒/mL(MDA-MB-231外泌体)、2.129×104个粒子/mL(SKBR3外泌体)和1.014×104颗粒/mL(MCF7外泌体)。
为了量化该检测方法的可靠性提高能力,我们对结果做了三种分析:
1、计算识别探针(红色)的像素点(单色检测方法):以CFPP的源代码为基础进行改写,通过遍历荧光图像中的所有像素点,并将R值大于等于200的像素点记为红色像素点,继而统计出荧光图片中红色像素点(即识别探针存在的位点)的个数。
2、计算识别探针和捕获探针(绿色)重合的像素点(即黄色的点)(双色检测方法):以CFPP的源代码为基础进行改写,通过遍历荧光图像中的所有像素点,并将R和G值大于等于200的像素点记为黄色像素点,继而统计出荧光图片中黄色像素点(即捕获探针和识别探针共同存在的位点)的个数。
3、计算捕获探针(绿色)、外泌体(蓝色)的识别探针(红色)重合的像素点(即白色点)(即前述的CFPP方法)。
结果如图6,(a)捕获探针和识别探针非特异性吸附导致的假阳性像素点(b)阳性像素点和外泌体或识别探针非特异性吸附导致的假阳性像素点。(c)三色荧光共定位策略、双色共定位策略以及常规单色荧光检测的CFPP结果(直方图)、双色共定位策略和常规单色荧光检测的假阳性率(折线图)。从图6可以看出,与双色共定位策略和传统的单色荧光检测相比,本发明的SR-TFC方法通过更严格的判断标准,实现了排除捕获探针、外泌体或识别探针的非特异性吸附引起的假阳性像素,更好地保证了特异性,提高了检测的可靠性。
实施例3
应用实施例2的方法分别检测MDA-MB-231、SKBR3和MCF7外泌体表面上的PD-L1、HER2和EpCAM三种乳腺癌特异性蛋白,将纯PBS缓冲液作为Control group。使用建立的CFPP,分析了共定位像素的数量,并在热图中对三种表面蛋白质进行了分析。热图结果如图5所示。可以看出,根据PD-L1、EpCAM和HER2的表达水平,三种外泌体群体都可以明显分离。将几组像素点个数统计如表1所示。
表1
HER2(像素点个数) | EpCAM(像素点个数) | PD-L1(像素点个数) | |
Control group | 13 | 54 | 92 |
MDA-MB-231 | 146 | 347 | 1055 |
MCF7 | 539 | 779 | 347 |
SKBR3 | 1571 | 755 | 522 |
从表1可以看出,HER2在SKBR3外泌体上高表达,在MCF7外泌体上中度表达,在MDA-MB-231外泌体上低表达。EpCAM在MCF7外泌体和SKBR3外泌体上呈现相似的含量。PD-L1在MDA-MB-231外泌体上高表达,在MCF7外泌体和SKBR3外泌体上呈现相似的低表达。因此,通过用SR-TFC和CFPP在外泌体上分析PD-L1、HER2和EpCAM,可以很容易地对不同类型的外泌体进行分类。
Claims (10)
1.一种面向高精度分析外泌体表型的超分辨率三色荧光共定位方法,其特征在于,包括如下步骤:
1)在制得的金纳米球阵列基底上修饰捕获探针;
2)在经过步骤1)处理后的金纳米球阵列基底上滴加外泌体溶液进行外泌体捕获;
3)在经过步骤2)处理后的金纳米球阵列基底上滴加膜染料,进行外泌体染色;
4)在经过步骤3)处理后的金纳米球阵列基底上滴加带有荧光的特异性识别探针进行特异性识别;
5)将样品放于单分子定位显微镜下对FAM通道、膜染料通道和Cy5 通道同时进行成像,激发光波长分别为488nm、561 nm和642nm,同时收集荧光信号并进行合成;
6)应用基于MATLAB的CFPP方法对步骤5)得到的三色超分辨图像进行共定位荧光像素点进行计数,所述CFPP方法是采用MATLAB的运行代码实现,以RGB色彩模式的原理为基础,在MATLAB设置像素点的R、G、B代表着颜色的三个基本分量,三个基本分量的取值范围均为0~255,不同的R、G、B组合会产生不同的颜色,CFPP通过遍历荧光图像中的所有像素点,并将R、G、B三个分量的值均大于等于200的像素点记为白色像素点,继而统计出荧光图片中白色像素点的个数即为特异性结合位点。
2.根据权利要求1所述的面向高精度分析外泌体表型的超分辨率三色荧光共定位方法,其特征在于,步骤1)中所述金纳米球阵列基底的制备方法如下:通过热蒸发在硅晶片上沉积铬层,通过气液界面加载技术,在所得的镀铬的硅片上紧密排列一层聚苯乙烯微球,通过热蒸发在所得的聚苯乙烯微球阵列的表面沉积一层金层。
3.根据权利要求2所述的面向高精度分析外泌体表型的超分辨率三色荧光共定位方法,其特征在于,所述气液界面加载技术具体步骤为:将聚苯乙烯微球溶液与无水乙醇混合,并超声处理,将混合溶液通过载玻片滑落到去离子水表面,在气液界面形成单层微球膜,滴加十二烷基硫酸钠溶液以固定微球膜,随后,将微球膜从水面转移到硅片上,并在室温下干燥,得到聚苯乙烯微球阵列。
4.根据权利要求2所述的面向高精度分析外泌体表型的超分辨率三色荧光共定位方法,其特征在于,所述铬层的厚度为15nm,所述金层的厚度为15nm。
5.根据权利要求3所述的面向高精度分析外泌体表型的超分辨率三色荧光共定位方法,其特征在于,所述聚苯乙烯微球溶液与无水乙醇以体积比2:1,所述聚苯乙烯微球溶液浓度为5wt%。
6.根据权利要求1所述的面向高精度分析外泌体表型的超分辨率三色荧光共定位方法,其特征在于,步骤1)中所述捕获探针为CD63适配体,其序列为5′-6FAM-CAC CCC ACCTCG CTC CCG TGA CAC TAA TGC TAT TTT TT-(CH2)6-SH-3′。
7.根据权利要求1所述的面向高精度分析外泌体表型的超分辨率三色荧光共定位方法,其特征在于,步骤2)中外泌体溶液包括MDA-MB-231、SKBR3和 MCF7外泌体溶液中的一种或几种。
8.根据权利要求7所述的面向高精度分析外泌体表型的超分辨率三色荧光共定位方法,其特征在于,步骤2)中MDA-MB-231外泌体溶液的浓度为7.124×104~7.124×107个/mL,所述SKBR3外泌体溶液的浓度为2.129×104 ~2.129×107个/mL,所述MCF7外泌体溶液浓度为1.014×104 ~1.014×107个/mL。
9.根据权利要求1所述的面向高精度分析外泌体表型的超分辨率三色荧光共定位方法,其特征在于,步骤4)中所述特异性识别探针包括Cy5-PD-L1适配体、Cy5-HER2适配体和Cy5-EpCAM适配体,Cy5-PD-L1适配体序列为:5′Cy5-TAC AGG TTC TGG GGG GTG GGT GGGGAA CCT GTT-3′;Cy5-HER2适配体序列为:5′Cy5-TTG GGC CGT CGA ACA CGA GCA TGG TGCGTG GAC CTA GGA TGA CCT GAG TAC TGT CCT-3′;
Cy5-EpCAM适配体序列为:5′Cy5-TTC ACT ACA GAG GTT GCG TCT GTC CCA CGT TGTCAT GGG GGG TTG GCC TGT-3′。
10.根据权利要求1~9任一项所述的面向高精度分析外泌体表型的超分辨率三色荧光共定位方法,其特征在于,所述捕获探针、外泌体、膜染料、特异性识别探针均溶解于PBS缓冲溶液中,所述PBS缓冲液pH=7.2-7.4,浓度为10 mM。
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