CN114152598A - 一种基于近红外光荧光寿命成像的细胞内细胞色素c的检测方法 - Google Patents
一种基于近红外光荧光寿命成像的细胞内细胞色素c的检测方法 Download PDFInfo
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Abstract
本发明实施例公开了一种基于近红外光荧光寿命成像的细胞内细胞色素c的检测方法,包括:将标记有核酸适配子的金纳米棒粒子溶液和标记有核酸适配子的近红外有机荧光分子溶液进行混合,离心,将得到的沉淀分散于缓冲溶液,得到检测剂,其中,近红外有机荧光分子上标记的核酸适配子为与细胞色素c特异性结合的核苷酸序列,金纳米棒粒子上标记的核酸适配子为与近红外有机分子上标记的核酸适配子可结合的核苷酸序列;将检测剂与细胞孵育混合至少20小时,待细胞摄取纳米检测体系后,采用近红外光激发,并测试近红外窗口内的荧光寿命;按照预先检测的荧光寿命与细胞色素c的含量关系得到细胞内细胞色素c的含量。
Description
技术领域
本发明实施例涉及化学技术领域,尤其是一种基于近红外荧光寿命成像的细胞内细胞色素c的检测方法。
背景技术
细胞色素c是一种生物生化反应常见的物质,特别是细胞早期凋亡过程的重要生物标志物。目前,细胞色素c的检查方法主要包含ELISA法、蛋白质印迹分析、质谱法、光学/电化学检测法等。尽管这些方法实现了细胞色素c的高灵敏度检测,但是无法直接进行细胞内的细胞色素c的检测。发展细胞内的细胞色素c的直接检测方法具有重大的生物检测意义。
近年来,核酸适配子技术的发展和纳米技术的发展为实现细胞内细胞色素c的检测提供了新的思路。核酸适配子可以和细胞色素c特异性结合,例如,采用纳米粒子结构输运核酸适配子结构到细胞内部,实现了细胞的荧光成像的细胞内的细胞色素c检测。然而细胞荧光成像的强度受多重因素干扰,如激发强度、光增益、光阑尺寸、有机荧光标记分子光漂白等因素,均可导致细胞强度的变化,从因影响细胞荧光成像检测的准确度。相对于,细胞荧光成像的缺点,细胞荧光寿命成像可以克服细胞荧光成像准确度低的缺点。荧光寿命是激发态荧光分子保持其激发态的平均时间。荧光寿命不会随细胞内荧光分子浓度、激发光功率变化而变化,因而相较于荧光强度更加准确可靠,尤其适应于细胞这种复杂的生理环境。
另一方面,紫外、可见光激发极易产生极强的背景荧光干扰,产生背景信号进一步降低检测灵敏度。而近红外光位于生物窗口内,因此,研发一种近红外激发的细胞荧光寿命成像检测细胞内细胞色素c的方法,成为人们亟待解决的问题。
发明内容
为解决上述技术问题,本发明创造的实施例提供一种基于近红外荧光寿命成像的细胞内细胞色素c的检测方法,包括:
将标记有核酸适配子的金纳米棒粒子溶液和标记有核酸适配子的近红外有机荧光分子溶液进行混合,离心,将得到的沉淀分散于缓冲溶液,得到检测剂,其中,近红外有机荧光分子上标记的核酸适配子为与细胞色素c特异性结合的核苷酸序列,金纳米棒粒子上标记的核酸适配子为与近红外有机分子上标记的核酸适配子可结合的核苷酸序列;
将检测剂与细胞孵育混合至少20小时,待细胞摄取纳米检测体系后,采用近红外光激发,并测试近红外窗口内的荧光寿命;
以预先设定的空白细胞荧光寿命为标准确定细胞内细胞色素c的变化。
优选地,金纳米棒粒子上标记的核酸适配子为5’端带有巯基的核苷酸序列。
优选地,带有巯基的核苷酸序列为-SH-TTTTTTGCAACAACG-。
优选地,近红外有机荧光分子上标记的核酸适配子为3’端修饰近红外有机荧光分子且可与癌胚抗原适配子特异性结合的核苷酸序列。
优选地,可与癌胚抗原适配子特异性结合的核苷酸序列为-ACCTGGGGGAGTATTGCGGAGGAAGGT-
优选的,标记有核酸适配子的金纳米棒与标记有核酸适配子的近红外有机荧光分子比为1:1000~1:50000,以保证两者的结合,有利于信号的稳定性与准确性。
更优选的,标记有核酸适配子的金纳米棒粒子与标记有核酸适配子的近红外有机荧光分子的摩尔比为1:5000,能保证金纳米棒和有机近红外分子的有效结合,从而保证信号的稳定性与准确性,也不造成原料的浪费,节约成本。
优选的,近红外荧光光谱的激发激光为波长为600~1000nm的近红外光,发射光荧光寿命探测为600~1000nm的近红外光。近红外荧光光谱的激发激光为近红外光,生物背景荧光低和组织穿透深。
更优选的,近红外荧光光谱的激发激光为波长为633nm的近红外光,发射光荧光寿命探测为650~800的近红外荧光寿命。
本发明采用构建近红外荧光寿命猝灭对的方式实现检测如图1所示,荧光寿命猝灭剂为金纳米棒,荧光寿命发光剂为近红外荧光分子(如Cy5.5荧光分子),两者通过核酸适配子进行结合,利用金纳米棒对近红外荧光分子的荧光寿命的猝灭,实现近红外有机分子的荧光寿命的猝灭。这种金纳米棒@近红外有机荧光分子对可以通过金纳米结构有效输运到细胞内。在细胞内部,所制备的复合物利用金纳米粒子保护特异性的核酸适配体子不被细胞酶代谢,并且细胞内的细胞色素c和复合物中的核酸适配子结合,导致近红外有机荧光分子的从金纳米棒表面解离,使近红外有机分子的荧光寿命增加,从而成功实现细胞内细胞色素c的检测。荧光寿命增加的越多,细胞色素c的含量越高,利用荧光寿命的变化判断细胞内细胞色素c的变化,具备检测结果稳定可靠、灵敏、噪声低等优点,实现活细胞内的细胞色素c的有效准确探测。
附图说明
为了更清楚地说明本发明实施例中的技术方案,下面将对实施例描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1是本发明一个检测原理示意图。
图2是本发明一个金纳米棒纳米粒子的近红外区域吸收光谱图和Cy5.5分子的近红外荧光发射光谱图。
图3是实施例1中未加入依托泊苷的空白对照组的633nm光激发下细胞荧光寿命成像图。
图4是实施例1中加入1μM依托泊苷的633nm光激发下细胞荧光寿命成像图。
图5是实施例1中加入2μM依托泊苷的633nm光激发下细胞荧光寿命成像图。
图6是实施例1中加入5μM依托泊苷的633nm光激发下细胞荧光寿命成像图。
具体实施方式
为了使本技术领域的人员更好地理解本发明方案,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述。
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
1)金纳米棒粒子的制备:将75μL HAuCl4(0.01M)添加到2.5mL CTAB(0.1M)中,形成均匀混合物。之后,在剧烈搅拌下加入200μL NaBH4(10mM)快速注入上述混合物中,立即获得金纳米种子溶液。之后配置金纳米棒生长溶液包含CTAB(0.1M,50mL)、AgNO3(0.01M,0.5mL)、HAuCl4(0.01M,2.5mL)和抗坏血酸(0.1M,0.375mL)。取金种子溶液0.5mL加入到上述生长液中,室温下反应2.5小时后,将溶液离心(1000rpm,15分钟。它重新分散在去离子水中。
2)金纳米棒@Cy5.5复合物制备:取步骤1)中金纳米棒粒子(约0.1nM,2mL),与200μL(500nM)巯基核酸适配子混合过夜后,10000rpm离心15min,离心两次去除过量未连接的核酸适配子,将离心后的沉淀重新分散稀释到2mL的Tris缓冲溶液中,其中,所述巯基核酸适配子,其序列如SEQ ID NO.1所示,5’端带有巯基修饰,即所述巯基核酸适配子的序列为5’-SH-TTTTTTGCAACAACG-3’(由生工生物工程(上海)股份有限公司合成),所述巯基核酸适配子与金三角纳米粒子通过巯基配位结合。取上述巯基适配子修饰的金纳米棒(约0.1nM,2mL),加入100μL 500nM的Cy5.5标记的细胞色素C适配子(其序列如SEQ ID NO.2所示,3’端带有Cy5.5分子修饰,核酸适配子的序列为5’-ACCTGGGGGAGTATTGCGGAGGAAGGT-Cy5.5-3’(由生工生物工程(上海)股份有限公司合成)),杂化混合4小时,离心半小时重新分散在2mLTris缓冲溶液中。
3)细胞内细胞色素c的检测:将六孔板(底部有盖玻片)接种4T1细胞(细胞密度2×105个/mL),在新鲜培养基中培养24小时,之后,加入步骤2)中的金纳米棒粒子@Cy5.5复合物(在细胞培养基浓度为0.025nM),孵育6小时之后,分为两组,第一组作为对照组,什么都不加,再在37℃下,孵育两小时后,在633nm近红外激光激发下,测试荧光寿命成像,细胞荧光成像图如图3所示,平均寿命为680ps。第二组,加入依托泊苷1μM在2mLDMEM中,在37℃下,孵育两小时后,在633nm近红外激光激发下,测试荧光寿命成像,细胞荧光成像图如图4所示,平均寿命为770ps。由于依托泊苷可以有效诱导细胞产出细胞色素c,由此可以细胞色素c的增加,导致Cy5.5修饰的细胞色素c适配子和细胞色素c连接而从金三角纳米粒子表面解离,从而市Cy5.5分子的荧光寿命增加,相对于控制组的寿命680ps增加到770ps。
实施例2
癌胚抗原的检测:将实施例1得到的金纳米棒粒子@Cy5.5复合物(在细胞培养基浓度为0.025nM),与细胞孵育孵育6小时(与实施例1步骤相同),加入依托泊苷2μM在2mL DMEM中,在37℃下,孵育两小时后,在633nm近红外激光激发下,测试荧光寿命成像,细胞荧光成像图如图5所示,平均寿命为820ps。由于依托泊苷可以有效诱导细胞产出细胞色素c,相对于实施例2加大了依托泊苷的剂量,由此可以诱导产生更多的细胞色素c,而Cy5.5分子的荧光寿命增加,平均寿命继续增加到820ps,从而可以判断细胞内细胞色素c大量产生。
实施例3
将实施例1得到的金纳米棒粒子@Cy5.5复合物(在细胞培养基浓度为0.025nM),与细胞孵育孵育6小时(与实施例1步骤相同),加入依托泊苷5μM在2mL DMEM中,在37℃下,孵育两小时后,在633nm近红外激光激发下,测试荧光寿命成像,细胞荧光成像图如图6所示,平均寿命为980ps。由于依托泊苷可以有效诱导细胞产出细胞色素c,相对于实施例3加大了依托泊苷的剂量,由此可以诱导产生更多的细胞色素c,而Cy5.5分子的荧光寿命增加,平均寿命继续增加到980ps,从而可以判断细胞内细胞色素c大量产生。
以上所述仅是本发明的部分实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种基于近红外光荧光寿命成像的细胞内细胞色素c的检测方法,其特征在于,包括:
将标记有核酸适配子的金纳米棒粒子溶液和标记有核酸适配子的近红外有机荧光分子溶液进行混合,离心,将得到的沉淀分散于缓冲溶液,得到检测剂,其中,近红外有机荧光分子上标记的核酸适配子为与细胞色素c特异性结合的核苷酸序列,金纳米棒粒子上标记的核酸适配子为与近红外有机分子上标记的核酸适配子可结合的核苷酸序列;
将检测剂与细胞孵育混合至少20小时,待细胞摄取纳米检测体系后,采用近红外光激发,并测试近红外窗口内的荧光寿命;
以预先设定的空白细胞荧光寿命为标准确定细胞内细胞色素c的变化。
2.根据权利要求1所述的检测方法,其特征在于,金纳米棒粒子上标记的核酸适配子为5’端带有巯基的核苷酸序列。
3.根据权利要求2所述的检测方法,其特征在于,带有巯基的核苷酸序列为-SH-TTTTTTGCAACAACG-。
4.根据权利要求1所述的检测方法,其特征在于,近红外有机荧光分子上标记的核酸适配子为3’端修饰近红外有机荧光分子且可与癌胚抗原适配子特异性结合的核苷酸序列。
5.根据权利要求4所述的检测方法,其特征在于,可与癌胚抗原适配子特异性结合的核苷酸序列为-ACCTGGGGGAGTATTGCGGAGGAAGGT-。
6.根据权利要求1所述的检测方法,其特征在于,标记有核酸适配子的金纳米棒粒子和标记有核酸适配子的近红外有机荧光分子的摩尔比为1:1000~50000。
7.根据权利要求1所述的检测方法,其特征在于,近红外荧光寿命的激发波长为600~1000nm。
8.根据权利要求1所述的检测方法,其特征在于,近红外光荧光寿命的测试波长为600-1000nm。
9.根据权利要求8所述的检测方法,其特征在于,近红外光荧光寿命的测试波长为633nm。
10.根据权利要求1所述的检测方法,其特征在于,近红外荧光寿命探测为650~800的近红外荧光寿命。
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