CN109239007A - 用于无标记检测细胞外泌体的功能化太赫兹狭缝纳米天线 - Google Patents
用于无标记检测细胞外泌体的功能化太赫兹狭缝纳米天线 Download PDFInfo
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Abstract
本发明涉及一种用于无标记检测细胞外泌体的功能化太赫兹狭缝纳米天线,包括基底以及溅镀在其表面的金属膜,所述基底包括双抛高阻硅以及在其表面氧化形成的二氧化硅层,金属膜上开设以二氧化硅层为底的若干个凹槽结构,所述凹槽结构的底部定向包被适配体序列,可特异结合外泌体表面靶分子,将外泌体固定于敏感响应区域。
Description
技术领域
本发明属于太赫兹测量技术领域,涉及用于无标记检测细胞外泌体的功能化太赫兹狭缝纳米天线。
背景技术
外泌体是由活细胞分泌的、直径在30-120nm的球形膜性囊泡,其内包含有蛋白质(CD9、CD63、CD81等跨膜蛋白超家族成员)、DNA、RNA(mRNA、微小RNA及非编码RNA)及脂质。外泌体广泛分布于外周血、唾液、腹水、脑脊液及尿液等体液中,参与细胞间信息传递、交换,在肿瘤发生、发展、耐药及组织损伤修复等多种生理或病理过程中发挥重要作用。不同细胞分泌的外泌体内容物存在差异,特别是其携带的特异蛋白和miRNA,可用于肿瘤的诊断、疗效评估及预后判断;对细胞分泌的外泌体进行准确定性和定量分析有望成为肿瘤液态活检新方法。然而,目前用于外泌体无标记定量分析的手段十分匮乏。
太赫兹(terahertz,THz)波位于微波和红外波之间,频率通常为0.1-10THz,具备应用于生物检测的独特优势,可以一种非电离方式探测生物大分子间包括范德华力、氢键等引起的分子间共振和偶极子旋转在内的微弱相互作用。然而太赫兹应用于生物分子检测仍面临分子吸收截面过小导致的灵敏度失匹配问题。太赫兹波段狭缝纳米天线是增强分子吸收、提高探测灵敏度的可靠途径。太赫兹狭缝纳米天线是由纳米级金膜经光刻形成亚波长周期性孔径结构,其基于超强光透射(Extraordinary Optical Transmission,EOT)现象,即当太赫兹波入射到狭缝天线阵列表面时,可为表面局域等离子体共振提供额外波矢量,当波矢完全匹配时,会出现耦合效率最高的透射谐振峰,从而极大增强狭缝内介质生物分子光谱吸收横截面,最终使介质分子的检测灵敏度提高103-1012倍;这为百纳米尺度级别细胞外泌体的无标记检测提供新思路。
但现有研究工作仅采用滴涂法使过量生物分子溶液渗入狭缝进行测量,而对于体液样本中痕量靶物质的测量,该方法显然无法满足要求。如何使待测痕量物质特异定位于电场极端增强的狭缝中并尽可能排除基质分子干扰是当前太赫兹狭缝纳米天线阵列需解决的关键问题。
发明内容
有鉴于此,本发明的目的在于提供一种用于无标记检测细胞外泌体的功能化太赫兹狭缝纳米天线。
为达到上述目的,本发明提供如下技术方案:
用于无标记检测细胞外泌体的功能化太赫兹狭缝纳米天线,包括基底以及溅镀在其表面的金属膜,所述基底包括双抛高阻硅以及在其表面氧化形成的二氧化硅层,金属膜上开设以二氧化硅层为底的周期排布的圆环形凹槽结构,圆环的外半径为10~80μm,环宽为100~2000nm,周期为20~200μm,所述圆环形凹槽结构的底部定向包被适配体序列,用于特异结合外泌体表面分子。
优选的,所述双抛高阻硅的厚度为500μm,电阻率大于10000Ω·cm。
优选的,所述二氧化硅层的厚度为200nm。
优选的,所述金属膜的材质选自金、铬、银、铜、铝或镍。
进一步优选的,所述金属膜包括上、下两层,分别为金膜和铬膜,厚度分别为200nm和20nm。
进一步优选的,所述凹槽结构的形状为圆环形,排布周期为120μm,外半径为40μm,内半径为39.5μm,缝隙为500nm。
优选的,通过共价偶联法将适配体序列定向包被于凹槽结构的底部。
进一步优选的,所述适配体序列为特异结合外泌体表面CD63分子,其序列如SEQID NO.1所示。
5’-CACCCCACCTCGCTCCCCGTGACACTAATGCTA-3’,SEQ ID NO.1。
上述功能化太赫兹狭缝纳米天线的制作方法,具体步骤如下:
(1)在双抛高阻硅的表面氧化出二氧化硅层,然后在二氧化硅层表面溅镀金属膜,在金属膜上开设以二氧化硅层为底的凹槽结构,得到太赫兹狭缝纳米天线;
(2)凹槽结构底部的二氧化硅层表面引入活性羟基基团,然后与γ-缩水甘油醚氧丙基三甲氧基硅烷(GOPS)中的硅基团反应生成硅烷化环氧基团,从而与适配体末端的磷酸根离子结合,使得适配体序列固定于凹槽结构底部的二氧化硅层表面。
优选的,步骤(1)中,采用电子束曝光技术加工凹槽结构。
优选的,步骤(2)中,利用等离子体清洗处理在凹槽结构底部的二氧化硅层表面引入活性羟基基团,具体方法是:使用美国Harrick等离子清洗机对凹槽结构底部的二氧化硅基底进行亲水羟基化处理,工作功率为100W,真空度0.3mBar,处理时间为12分钟。
优选的,步骤(2)中,硅烷化环氧基团的生成方法为:引入活性羟基基团后的太赫兹狭缝纳米天线转移至含0.001mol/L GOPS的丙酮溶液中,室温(25℃)反应30分钟后取出,清洗,烘干即可。
进一步优选的,引入活性羟基基团后的太赫兹狭缝纳米天线先放入超纯水中浸泡15分钟,用氮气吹干后再利用GOPS的丙酮溶液处理。
进一步优选的,利用丙酮和无水乙醇依次清洗,每种溶剂的清洗次数均为3次。
优选的,步骤(2)中,适配体序列的固定方法如下:将生成硅烷化环氧基团的太赫兹狭缝纳米天线放入反应适配体溶液中,氮气保护下,50℃反应2小时,取出后清洗即可。
进一步优选的,所述反应适配体溶液是利用pH=10.0的反应缓冲液将适配体序列储存液稀释为10μmol/L,其中,反应缓冲液的组成如下:含10μL 1-甲基咪唑、125.5μL二甲基亚砜及1119μL超纯水;适配体序列储存液的浓度为100μmol/L,还含有:10mM Tris-HCl,100mM NaCl,10mM MgCl2,PH=8.0。
进一步优选的,利用pH=7.0的清洗缓冲液和超纯水依次清洗。
更进一步优选的,所述清洗缓冲液的组成如下:50mL超纯水中含有438mg氯化钠,205.5mg柠檬酸二钠,使用1mol/L盐酸调整PH。
本发明的有益效果在于:
本发明针对现有细胞外泌体定量分析手段匮乏的状况,引入圆环形太赫兹狭缝纳米天线进行高灵敏探测;并针对现有太赫兹超材料增强检测的滴涂加样法所面临样本随机分布造成的灵敏度和特异度低的难题,创新性将待检痕量物质(外泌体)特异定位于狭缝纳米天线增强电场处(二氧化硅基底面)以实现高灵敏响应,具体通过构建圆环形狭缝纳米天线,使其具备对入射THz波极化不敏感,极大方便偏振THz光谱检测过程;将适配体序列(靶向结合肿瘤细胞外泌体高度表达的跨膜蛋白CD63分子)定向包被在圆环狭缝处二氧化硅基底上,可靶向捕获外泌体并固定于圆环狭缝处,进而通过狭缝处增强的表面等离子体共振极大提升外泌体的吸收截面,实现对外泌体的高灵敏、无标记检测。同时,该定向包被亲和性物质于狭缝电场增强处的思路亦可用于其他分子的高灵敏检测。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚,本发明提供如下附图进行说明:
图1为太赫兹圆环形狭缝纳米天线阵列检测示意图;
图2为FDTD模拟分析圆环形狭缝纳米天线共振电场分布(x-y轴);
图3为太赫兹圆环形狭缝纳米天线对梯度浓度MCF-7外泌体溶液响应的归一化反射谱图;
图4为MCF-7外泌体溶液在太赫兹圆环形狭缝纳米天线频移响应与浓度的线性拟合图;
其中,1为金属膜,2为二氧化硅层,3为双抛高阻硅,4为外泌体,5为适配体。
具体实施方式
下面将结合附图,对本发明的优选实施例进行详细的描述。
实施例:
如图1所示,用于无标记检测细胞外泌体的功能化太赫兹狭缝纳米天线,包括基底以及溅镀在其表面的金属膜1,基底包括双抛高阻硅3以及在其表面氧化形成的二氧化硅层2,金属膜1上开设以二氧化硅层2为底的若干个凹槽结构,凹槽结构的底部定向包被适配体5序列,用于特异结合外泌体4的表面分子。
双抛高阻硅3的厚度为500μm,电阻率大于10000Ω·cm。二氧化硅层2的厚度为200nm。金属膜1包括上、下两层,分别为金膜和铬膜,厚度分别为200nm和20nm。
凹槽结构的形状为圆环形,分布周期为120μm,外半径为40μm,内半径为39.5μm,缝隙为500nm。
通过共价偶联法将适配体5序列定向包被于凹槽结构的底部。适配体5序列为特异结合外泌体表面CD63分子,其序列如SEQ ID NO.1所示。
5’-CACCCCACCTCGCTCCCCGTGACACTAATGCTA-3’,SEQ ID NO.1。
上述功能化太赫兹狭缝纳米天线的制作方法,具体步骤如下:
(1)在双抛高阻硅3的表面氧化出二氧化硅层2,然后在二氧化硅层2表面溅镀金属膜,1采用电子束曝光技术在金属膜1上开设以二氧化硅层2为底的凹槽结构,得到太赫兹狭缝纳米天线;
(2)凹槽结构底部的二氧化硅层2表面引入活性羟基基团,然后与γ-缩水甘油醚氧丙基三甲氧基硅烷(GOPS)中的硅基团反应生成硅烷化环氧基团,从而与适配体5末端的磷酸根离子结合,使得适配体5序列固定于凹槽结构底部的二氧化硅层2表面;具体方法如下:
(2-1)狭缝纳米芯片的硅烷化:
使用美国Harrick等离子清洗机对太赫兹圆形狭缝纳米天线的二氧化硅基底进行亲水羟基化处理,设定工作功率为100W,真空度0.3mBar,处理时间为12分钟。等离子体清洗结束后,将芯片取出并放入超纯水中浸泡15分钟,用氮气将玻片吹干后,将芯片转移入浓度为0.001mol/L的GOPS的丙酮溶液中,室温下反应半个小时后取出,用丙酮和无水乙醇依次清洗3次后,将芯片烘干备用。此时已实现对芯片狭缝处二氧化硅基底的硅烷化修饰。
(2-2)适配体的固定:
取出100μmol/L适配体序列储存液(10mM Tris-HCl,100mM NaCl,10mM MgCl2,PH=8.0),配置PH为10.0的反应缓冲液(含10μL 1-甲基咪唑、125.5μL二甲基亚砜及1119μL超纯水)及PH为7.0的清洗缓冲液(50mL超纯水中含有438mg氯化钠,205.5mg柠檬酸二钠,使用1mol/L盐酸调整PH)。用反应缓冲液将适配体序列稀释为10μmol/L的反应适配体溶液,并将上述硅烷化的狭缝纳米芯片放入其中,在氮气保护下、温度为50℃反应2个小时,取出芯片后用清洗缓冲液和超纯水彻底冲洗2次,以形成功能化太赫兹狭缝纳米芯片。
由FDTD(时域有限差分分析)模拟所得图2可得,在偏振太赫兹波正入射的情况下,可激发狭缝天线的明偶极子模式(TE11模式),增强的共振电场集中分布于圆环狭缝内部。
其中,f为圆环狭缝纳米天线的共振频率,c为光速,r为圆环的外半径,neff为等效折射率。
由公式(1)可得,圆环狭缝纳米天线的共振频率f在其周长确定情况下,与狭缝附近等效折射率相关;当待测靶物质进入狭缝内部时,导致其等效折射率升高(以空气为参考),使共振频率降低,发生红移,以此可对待测物质进行定量分析。
试验例
乳腺癌细胞外泌体的检测:
使用体积百分数10%无外泌体(exosome-free)的胎牛血清、链霉素(100μg/mL)及青霉素(100U/mL)配置DMEM培养基,在37℃、二氧化碳浓度为5%的环境下培养人乳腺癌MCF-7细胞(HTB-22TM)。采用标准的超速离心法获取MCF-7细胞分泌的外泌体,具体为:取出传代培养后48小时、生长状态良好的细胞培养基,在4℃条件下300g离心10分钟,获取上清液后,再于2000g离心20分钟;将上清液经0.22μm滤膜过滤后,在4℃条件下100000g超速离心75分钟;除去上清液后,使用PBS缓冲液洗涤沉淀并重悬后,于4℃条件下100000g超速离心75分钟,经PBS缓冲液(PH为7.4,含135mM NaCl,4.7mM KCl,10mM Na2HPO4,2mMNaH2PO4)洗涤沉淀并重悬后即获得MCF-7细胞外泌体溶液。
纯化后外泌体溶液由纳米颗粒跟踪分析仪(NTA)测定其水合半径及浓度,并通过梯度稀释配置浓度为1×103、1×104、1×105、1×106、1×107个/μL的外泌体溶液。滴加20μL对应浓度的外泌体溶液于功能化太赫兹狭缝纳米芯片表面,并均匀分散开。在室温下反应30分钟后去除表面的反应溶液,用PBS缓冲液冲洗两次后,再用超纯水快速冲去PBS残余液,轻柔氮气吹干后检测。使用反射式太赫兹光谱仪(光路空气湿度在3%以下),平均2048次采集光谱,在反射时域谱上截取携带外泌体响应信号的第二个反射峰进行快速傅里叶变换,获得频域强度值。同样方法检测不同梯度浓度的外泌体悬液及空白功能化的太赫兹纳米天线阵列。将空白功能化太赫兹纳米天线及梯度浓度外泌体悬液的频域强度值与对应空白硅片的强度值相除,并将反射谱图进行归一化处理,如图3所示。
随着外泌体悬液浓度升高,圆环形狭缝纳米天线的共振频率逐渐向低频波段发生移动,并随着浓度升高,频移值依次增大。MCF-7细胞的外泌体上的CD63跨膜蛋白可以与狭缝基底上适配体序列发生特异结合,从而将外泌体固定于狭缝内部。故随着外泌体浓度升高,狭缝内部捕获的数量随之增多,对应的圆环狭缝处等效介电常数会升高,导致圆环形纳米天线的共振频率发生红移。提取梯度浓度外泌体悬液在太赫兹纳米天线上的频率位移值,与其对应浓度的对数值进行线性拟合,如图4所示,线性拟合度较好,说明构建的功能化圆环形狭缝纳米天线可以通过共振频率位移实现对悬液中肿瘤细胞外泌体的高灵敏无标记定量分析。
最后说明的是,以上优选实施例仅用以说明本发明的技术方案而非限制,尽管通过上述优选实施例已经对本发明进行了详细的描述,但本领域技术人员应当理解,可以在形式上和细节上对其作出各种各样的改变,而不偏离本发明权利要求书所限定的范围。
序列表
<120> 用于无标记检测细胞外泌体的功能化太赫兹狭缝纳米天线
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
caccccacct cgctccccgt gacactaatg cta 33
Claims (7)
1.用于无标记检测细胞外泌体的功能化太赫兹狭缝纳米天线,其特征在于,包括基底以及溅镀在其表面的金属膜,所述基底包括双抛高阻硅以及在其表面氧化形成的二氧化硅层,金属膜上开设以二氧化硅层为底的周期排布的圆环形凹槽结构,圆环的外半径为10~80μm,环宽为100~2000nm,周期为20~200μm,所述圆环形凹槽结构的底部定向包被适配体序列,用于特异结合外泌体表面分子。
2.根据权利要求1所述的功能化太赫兹狭缝纳米天线,其特征在于,通过共价偶联法将适配体序列定向包被于凹槽结构的底部。
3.权利要求1或2所述功能化太赫兹狭缝纳米天线的制作方法,其特征在于,具体步骤如下:
(1)在双抛高阻硅的表面氧化出二氧化硅层,然后在二氧化硅层表面溅镀金属膜,在金属膜上开设以二氧化硅层为底的凹槽结构,得到太赫兹狭缝纳米天线;
(2)凹槽结构底部的二氧化硅层表面引入活性羟基基团,然后与γ-缩水甘油醚氧丙基三甲氧基硅烷中的硅基团反应生成硅烷化环氧基团,从而与适配体末端的磷酸根离子结合,使得适配体序列固定于凹槽结构底部的二氧化硅层表面。
4.根据权利要求3所述的制作方法,其特征在于,步骤(1)中,采用电子束曝光技术加工凹槽结构。
5.根据权利要求3所述的制作方法,其特征在于,步骤(2)中,利用等离子体清洗处理在凹槽结构底部的二氧化硅层表面引入活性羟基基团,具体方法是:使用等离子清洗机对凹槽结构底部的二氧化硅基底进行亲水羟基化处理,工作功率为100W,真空度0.3mBar,处理时间为12分钟。
6.根据权利要求3所述的制作方法,其特征在于,步骤(2)中,硅烷化环氧基团的生成方法为:引入活性羟基基团后的太赫兹狭缝纳米天线转移至含0.001mol/L GOPS的丙酮溶液中,室温反应30分钟后取出,清洗,烘干,即可。
7.根据权利要求3所述的制作方法,其特征在于,步骤(2)中,适配体序列的固定方法如下:将生成硅烷化环氧基团的太赫兹狭缝纳米天线放入反应适配体溶液中,氮气保护下,50℃反应2小时,取出后清洗即可。
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110031423A (zh) * | 2019-05-23 | 2019-07-19 | 中国人民解放军陆军军医大学第一附属医院 | 基于太赫兹波技术的无标记评估水通道蛋白功能的方法 |
CN110132881A (zh) * | 2019-02-02 | 2019-08-16 | 中国人民解放军陆军军医大学第一附属医院 | 集成太赫兹和拉曼光谱的多光谱纳米阵列芯片和应用 |
CN113088565A (zh) * | 2021-04-26 | 2021-07-09 | 中国人民解放军陆军军医大学第一附属医院 | 一种快速检测microRNA的太赫兹芯片及其检测方法 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100278697A1 (en) * | 2004-08-04 | 2010-11-04 | Tae-Woong Koo | Methods and systems for detecting biomolecular binding using terahertz radiation |
US20150280036A1 (en) * | 2013-03-15 | 2015-10-01 | Wright State University | THz DISTRIBUTED DETECTORS AND ARRAYS |
CN104977272A (zh) * | 2015-07-17 | 2015-10-14 | 浙江大学 | 太赫兹超材料与纳米金颗粒联用的生物样品信号放大方法 |
CN105588820A (zh) * | 2015-12-15 | 2016-05-18 | 中国人民解放军第三军医大学第一附属医院 | 基于太赫兹超材料检测微量活细菌的方法 |
CN107446807A (zh) * | 2017-07-26 | 2017-12-08 | 中国人民解放军第三军医大学第附属医院 | 集成式太赫兹超结构纳米生物芯片及其应用和方法 |
-
2018
- 2018-10-29 CN CN201811271530.4A patent/CN109239007B/zh active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20100278697A1 (en) * | 2004-08-04 | 2010-11-04 | Tae-Woong Koo | Methods and systems for detecting biomolecular binding using terahertz radiation |
US20150280036A1 (en) * | 2013-03-15 | 2015-10-01 | Wright State University | THz DISTRIBUTED DETECTORS AND ARRAYS |
CN104977272A (zh) * | 2015-07-17 | 2015-10-14 | 浙江大学 | 太赫兹超材料与纳米金颗粒联用的生物样品信号放大方法 |
CN105588820A (zh) * | 2015-12-15 | 2016-05-18 | 中国人民解放军第三军医大学第一附属医院 | 基于太赫兹超材料检测微量活细菌的方法 |
CN107446807A (zh) * | 2017-07-26 | 2017-12-08 | 中国人民解放军第三军医大学第附属医院 | 集成式太赫兹超结构纳米生物芯片及其应用和方法 |
Non-Patent Citations (2)
Title |
---|
JIE SHU等: "Extraordinary THz Transmission in Ring Apertures", 《2011 INTERNATIONAL CONFERENCE ON INFRARED MILLIMETER, AND TERAHERTZ WAVES》 * |
LIJUAN XIE等: "Extraordinary sensitivity enhancement by metasurfaces in terahertz detection of antibiotics", 《SCIENTIFIC REPORTS》 * |
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CN110132881A (zh) * | 2019-02-02 | 2019-08-16 | 中国人民解放军陆军军医大学第一附属医院 | 集成太赫兹和拉曼光谱的多光谱纳米阵列芯片和应用 |
CN110031423A (zh) * | 2019-05-23 | 2019-07-19 | 中国人民解放军陆军军医大学第一附属医院 | 基于太赫兹波技术的无标记评估水通道蛋白功能的方法 |
CN113088565A (zh) * | 2021-04-26 | 2021-07-09 | 中国人民解放军陆军军医大学第一附属医院 | 一种快速检测microRNA的太赫兹芯片及其检测方法 |
CN113088565B (zh) * | 2021-04-26 | 2023-07-14 | 中国人民解放军陆军军医大学第一附属医院 | 一种快速检测microRNA的太赫兹芯片及其检测方法 |
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