CN114130438A - 一种分泌型自噬小体表面蛋白检测芯片制备方法及应用 - Google Patents
一种分泌型自噬小体表面蛋白检测芯片制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种分泌型自噬小体表面蛋白检测芯片制备方法及应用,芯片的制备方法如下:根据设计的掩模版,采用软光刻法制备梭形阵列微流控芯片,将生物素化的明胶溶液、亲和素溶液依次交替通入至芯片内并静置,从而实现芯片表面的层层组装;在经过组装后的芯片表面修饰生物素化的捕获抗体LC3B,即可得到微流控芯片;本发明还制备了不同量子点修饰的检测抗体探针用于分泌型自噬小体表面蛋白检测;本发明具有样本消耗量少、检测快速简便、选择性好、灵敏度高,高通量,稳定性好等优点,可达到相关癌症诊断的目的。
Description
技术领域
本发明属于微流控芯片制备方法及用途,特别涉及一种分泌型自噬小体表面蛋白检测芯片制备方法及应用。
背景技术
2016年10月,日本科学家Yoshinori Ohsumi因揭示“自噬机制”获得诺贝尔生理学或医学奖,自此,“自噬”再次成为热门话题。研究表明自噬在肿瘤的生长和转移中起重要作用,它通常被描述为分解代谢,但最新研究发现胞内形成的自噬小体可以释放到细胞外基质中。这种自噬小体被称为分泌型自噬小体,是细胞外囊泡的一种,尺寸约为500nm,具有典型的双层膜结构,表达自噬小体特异性标志蛋白LC3B,并携带来自母细胞的信号分子和表达蛋白,可作为癌症早期检测的生物标志物。正常生理条件下,细胞自噬维持在较低的基础水平,然而,在氧气及营养缺乏的肿瘤微环境中,自噬水平会显著升高。因此,通过研究肿瘤微环境中自噬小体的分泌规律、分泌量及膜表面蛋白的表达,有望达到肿瘤诊断的目的。
细胞外囊泡的传统分离检测方法较多,如超速离心法、免疫亲和捕获法、尺寸梯度过滤法等。这些方法较为复杂,耗时较长,且难以同时保证分泌型自噬小体的生物学活性与高纯度,难以满足临床大样本分离检测的要求。而微流控芯片是最实用的解决方案之一,因为它们可以同时分离和检测目标,具有微型化、高通量、试剂用量低、易于与其他设备集成等优势,被广泛应用于生化检测、化学合成、医学研究、司法鉴定等领域。在液体活检中,利用微流控新技术,结合纳米科学、临床医学等领域的新技术新方法,发展液体活检新技术,开发自噬小体临床检测芯片,不仅可以提高疾病早期诊断率,还在极大程度上减轻了病患身体上的疼痛。而进一步深入研究自噬小体的生物学结构及功能,在指导疾病早期诊断及临床药物研究方面均提供了新思路。
现有的自噬小体研究方法主要适用于降解型自噬小体,研究环境多为细胞内追踪检测,且无法在体外高通量、高灵敏检测自噬小体表面多种蛋白的问题。
发明内容
发明目的:本发明提供一种快速、准确、高灵敏的用于自噬小体表面蛋白检测的微流控芯片制备方法。
本发明的另一目的是提供所述微流控芯片的用途。
技术方案:本发明的分泌型自噬小体表面蛋白检测芯片制备方法,步骤如下:
(1)制备带有检测通道的梭形阵列化芯片,采用等离子体清洗机将玻璃片与PDMS芯片键合;
(2)将生物素化的明胶溶液、亲和素溶液依次交替通入步骤(1)制备的芯片内,对芯片表面进行层层组装;
(3)在经过组装后的芯片表面修饰生物素化的捕获抗体LC3B,随后PBS溶液冲洗,即可得到微流控芯片。
进一步地,所述步骤(2)中生物素化明胶溶液质量浓度为0.1~1%,亲和素溶液浓度为50~200μg/mL。生物素化明胶的制备工艺如下:向明胶溶液中加入Sulfo-NHS-biotin,搅拌,透析,冷冻、干燥,即可得到生物素化明胶。所述明胶与Sulfo-NHS-biotin的质量比为4~10∶1。其中,所述微流控芯片通道为梭形阵列,用于分泌型自噬小体表面蛋白检测。所述梭形阵列化芯片尺寸优选长为125μm、宽为31μm、高为50μm,每相邻两个阵列之间的间隔为47μm。生物素化明胶优选4层。
所述步骤(3)中捕获抗体LC3B的浓度为10~60μg/mL。
一种利用所述方法制备的微流控芯片在自噬小体表面蛋白检测中的用途。
进一步地,所述检测方法如下:
(1)将自噬小体通入芯片内,室温孵育,然后用PBST溶液冲洗;
(2)随后加入脱脂牛奶处理步骤(1)所得芯片,然后用PBS溶液洗涤;
(3)加入量子点偶联的检测抗体探针混合液,室温下反应,随后PBS溶液洗涤,并排尽液体晾干;
(4)对步骤(3)所得样品进行荧光成像,最后通过软件分析荧光强度得到不同标志物的表达情况。
进一步地,所述量子点偶联的检测抗体探针混合液的制备方法如下:
(1)羧基的活化:取量子点溶液,EDC溶液,NHS溶液,加入到PBS缓冲溶液中,室温震荡;
(2)配制Na2CO3溶液,向(1)反应液中加入Na2CO3溶液,调节pH;
(3)向上述步骤(2)反应液中加入待标记的抗体LC3B,室温震荡;充分混匀后将反应液冷藏;
(4)待步骤(2)反应结束后,将溶液离心,除去多余杂质,最后将产物分散在PBS缓冲溶液中,冷藏。
有益效果:本发明与现有技术相比,具有如下优势:
1、本发明首次提出对分泌型自噬小体胞外分离收集检测。
2、本发明方法所需样品量少,无需样品前处理,耗时短,分离效率高。
3、本发明方法可以同时快速检测分泌型自噬小体表面多种蛋白标志物,减少了检测次数,同时对样品的需求量减少了,其操作步骤简单。
4、本发明具有样本消耗量少、检测快速简便、选择性好、灵敏度高,高通量等优点。
附图说明
图1为基于微流控芯片检测分泌型自噬小体表面蛋白的原理图;
图2A为芯片内梭形阵列分布的光学显微镜图;图2B显示了荧光标记明胶膜的共聚焦成像;图2C为梭形阵列分布及尺寸的SEM图像;图2D为阵列芯片荧光标记的平面扫描以及3D图像,其中,图2a为芯片实物图;
图3A为自噬小体的TEM图,标尺500纳米;图3B为囊泡粒径分布;图3C为自噬小体的表面蛋白Western blot验证图;图3D为自噬小体表面特征蛋白流式图;
图4A为阵列芯片检测分泌型自噬小体表面蛋白HSP60的标准曲线;图4B为阵列芯片检测分泌型自噬小体表面蛋白LC3B的校准曲线;图4C为在不同浓度分泌型自噬小体样本中,QD1-HSP60信号强度被QD2-LC3B归一化后的图像;
图5A为等量相同样品分别加入等体积PBS溶液和去除囊泡的临床血清溶液中后,检测分泌型自噬小体表面LC3B蛋白的图象对比;图5B为卵巢癌细胞SKOV3和正常细胞IOSE80上清中分泌型自噬小体表面LC3B和HSP60蛋白的水平;图5C为卵巢癌患者与正常人分泌型自噬小体表面HSP60蛋白表达的区别图像。
具体实施方式
本发明基于明胶膜修饰的微流控芯片实现血清中自噬小体表面蛋白检测的实施过程如图1所示。
第一步,梭形阵列芯片制作
本发明微流控芯片的通道图形采用AutoCAD(AutoCAD 2020,Autodesk Inc.)软件自行绘制。掩模板由中心启恒制备。利用PDMS和玻璃结合共同制作微流控芯片,该芯片长为125μm、宽为31μm、高为50μm,每相邻两个阵列之间的间隔为47μm。
第二步,芯片内明胶膜修饰
首先制备生物素化明胶,在搅拌条件下,以3.5/1的质量比向4%(w/v)明胶中添加Sulfo-NHS-biotin。调整pH值为7.4,室温下反应过夜。使用10k Mw透析袋透析生物素明胶48h。透析后,冷冻干燥1天,4℃保存备用。
采用逐层组装技术对微流控芯片进行表面修饰:PDMS通过氧等离子体活化与玻璃片结合后,将1%(w/v)生物素明胶注入通道,室温下孵育15min,随后通入100μg/mL亲和素交联剂孵育15min,重复3次,得到4层明胶膜。注意每交替通入溶液前,用PBS清洗3次。
第三步,芯片内抗体修饰
对经过明胶膜修饰后芯片的明胶膜表面,加入50μg/mL生物素化的捕获抗体LC3B,并在通道孵育1h,PBST洗涤3次后,4℃保存备用。
第四步,检测抗体与量子点偶联
(1)羧基的活化:取5μL量子点(5μM)溶液,2μL EDC溶液(16mM),1μL NHS溶液(10mM),加入到50μL PBS缓冲溶液(10mM,pH 7.4)中,室温震荡15min;
(2)配制Na2CO3溶液,向(1)反应液中加入Na2CO3溶液,将pH调至9.0左右;
(3)向上述反应液中加入待标记的抗体LC3B,室温震荡30min;
(4)充分混匀后将反应液放置冰箱4℃过夜;
(5)待反应结束后,将溶液离心,除去多余杂质,PBS洗涤三次,最后将产物分散在PBS缓冲溶液中,放置4℃存放备用。
第五步,芯片内自噬小体捕获及检测
向通道中通入2μL收集的血清样本,室温静置反应2h,PBS冲洗5min,随后,通道内加入2μL两种量子点标记的抗体混合液,室温反应1h。激光共聚焦显微镜成像,测定血清样本中卵巢癌相关自噬小体表面蛋白的表达情况,实现卵巢癌的准确诊断。
以下是对各图的解释说明:
(1)附图1为蛋白检测芯片制备原理图,展示了PDMS芯片通道包含垂直交错均匀分布的梭型微柱阵列,通过生物化明胶和亲和素逐层组装和修饰,利用生物素-亲和素的特异性作用,固定自噬小体表面特异性抗体,卵巢癌细胞SKOV-3/卵巢上皮细胞IOSE80的自噬小体通过芯片时被抗体抓捕,随后用LC3B和HSP60两种抗体分别标记两个激发相同、发射波长不同的量子点,同时检测多种自噬小体蛋白,通过蛋白表达水平的不同来达到诊断的目的。
(2)对制作的芯片进行了显微镜放大表征,荧光分子修饰的荧光共聚焦表征,SEM表征和3D荧光共聚焦表征,结果如图2所示,可以看出芯片尺寸符合我们的设计,并且芯片表面明胶膜在通道中成功组装,且均匀分布。
(3)对我们离心收集的自噬小体使用透射电镜(TEM)、纳米颗粒跟踪分析(NTA)、蛋白质免疫印迹(Western blot)和流式细胞仪技术进行表征鉴定。结果如图3所示,透射电镜观察发现自噬小体是一个囊泡,大小约为500nm,为双层膜结构。NTA进一步检测验证了TEM的结果,平均粒径达到436.3nm。Western blot结果显示,收集到的自噬小体表达特异性蛋白LC3B。流式细胞术发现,超过95%的TRAP膜表面表达自噬小体LC3B的特征标记物,验证了Western blot的结果。因此我们所收集的样品确实是自噬小体。
(4)选取卵巢癌细胞SKOV3来源的自噬小体作为标准品,利用蛋白检测芯片检测自噬小体表面的不同蛋白LC3B和HSP60。从实验结果图4可以得到,在10-105μL-1和10-107μL-1范围内,两种探针的信号强度都随着自噬体浓度的增加而增加。QD1 HSP60探针的检出限(LOD)为126μL-1。由于LC3B是自噬体的通用标记物,可以通过其信号归一化处理来计算自噬体靶生物标志物的相对表达量,如图3C所示,LC3B的归一化HSP60信号并没有随着芯片中自噬小体浓度的增加而增加,其斜率也没有明显波动,基本为零。这一结果表明,LC3B一方面可以反映自噬小体的数量,另一方面可以根据自噬小体生物标志物的归一化信号反映样品中HSP60的丰度。
(5)本芯片对于实际临床样本的应用探究如图5所示,表明临床血清样本对本芯片检测不会产生影响,且证实了癌症细胞和正常细胞在LC3B和HSP60蛋白的表达上确实存在差异,为此芯片的应用奠定了基础。对于临床样本确实能达到区分癌症患者和正常人的目的。
Claims (8)
1.一种分泌型自噬小体表面蛋白检测芯片制备方法,其特征在于:步骤如下:
(1)制备带有检测通道的梭形阵列化芯片,采用等离子体清洗机将玻璃片与PDMS芯片键合;
(2)将生物素化的明胶溶液、亲和素溶液依次交替通入步骤(1)制备的芯片内,对芯片表面进行层层组装;
(3)在经过组装后的芯片表面修饰生物素化的捕获抗体LC3B,随后PBS溶液冲洗,即可得到微流控芯片。
2.根据权利要求1所述的分泌型自噬小体表面蛋白检测芯片制备方法,其特征在于:所述步骤(2)中生物素化明胶溶液质量浓度为0.1~1%,亲和素溶液浓度为50~200μg/mL。
3.根据权利要求1所述的分泌型自噬小体表面蛋白检测芯片制备方法,其特征在于:所述步骤(2)生物素化明胶的制备工艺如下:向明胶溶液中加入Sulfo-NHS-biotin,搅拌,透析,冷冻、干燥,即可得到生物素化明胶。
4.根据权利要求4所述的分泌型自噬小体表面蛋白检测芯片制备方法,其特征在于:所述明胶与Sulfo-NHS-biotin的质量比为4~10∶1。
5.根据权利要求1所述的自噬小体表面蛋白检测的微流控芯片制备方法,其特征在于:所述步骤(3)中捕获抗体LC3B的浓度为10~60μg/mL。
6.一种利用权利要求1-5任一项所述方法制备的微流控芯片在自噬小体表面蛋白检测中的用途。
7.根据权利要求6所述的用途,其特征在于:所述检测方法如下:
(1)将自噬小体通入芯片内,室温孵育,然后用PBST溶液冲洗;
(2)随后加入脱脂牛奶处理步骤(1)所得芯片,然后用PBS溶液洗涤;
(3)加入量子点偶联的检测抗体探针混合液,室温下反应,随后PBS溶液洗涤,并排尽液体晾干;
(4)对步骤(3)所得样品进行荧光成像,最后通过软件分析荧光强度得到不同标志物的表达情况。
8.根据权利要求7所述的用途,其特征在于:所述量子点偶联的检测抗体探针混合液的制备方法如下:
(1)羧基的活化:取量子点溶液,EDC溶液,NHS溶液,加入到PBS缓冲溶液中,室温震荡;
(2)配制Na2CO3溶液,向(1)反应液中加入Na2CO3溶液,调节pH;
(3)向上述步骤(2)反应液中加入待标记的抗体LC3B,室温震荡;充分混匀后将反应液冷藏;
(4)待步骤(2)反应结束后,将溶液离心,除去多余杂质,最后将产物分散在PBS缓冲溶液中,冷藏。
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