CN114487066A - 一种超灵敏dna-生物大分子传感器、构建及其应用 - Google Patents
一种超灵敏dna-生物大分子传感器、构建及其应用 Download PDFInfo
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Abstract
本发明提供一种超灵敏DNA‑生物大分子传感器,构建及其应用。系由溶液相、界面相以及经辅助探针自组装的金电极组成,溶液相由适配体/信号探针双链组成,界面相为上述的金电极和溶液相之间的界面。本发明还提供了上述传感器在癌症样本中的检测应用,能有效的检测乳腺癌、卵巢癌和前列腺癌血浆样本中的促黄体生成激素。本发明传感器制备简便,成本低廉,可重复使用。
Description
技术领域
本发明属于生物技术和检测技术领域,具体涉及一种超灵敏DNA-生物大分子传感器、构建及其在临床肿瘤样本中的检测应用。
背景技术
生物大分子(如激素和蛋白质)的体外诊断检测,对疾病和癌症的早期预警起着至关重要的作用。目前生物大分子检测的主流方法是免疫印迹法(western blots,WB)和酶联免疫吸附试验(ELISA),它们具有较高的灵敏度和选择性,但也存在仪器和试剂昂贵、多步骤、再生困难等问题。随着对卫生概念的日益重视,迫切需要开发成本低廉、操作简单和灵敏度高的检测平台,以便满足频繁和即时的诊断应用。
近年来,随着配体指数富集系统进化(SELEX)技术的不断发展,基于核酸适配体的电化学(E-AB)传感器有望成为低成本、简单、灵敏的即时诊断系统。例如,Kevin W.Plaxco及其同事开发了一种E-AB传感器,可直接检测血清中血小板衍生生长因子,检测限为50pM。此外,他们还证明了E-AB传感器可以连续、实时地测量活体动物的药代动力学。最近,Tanner等通过SELEX技术获得了一个高亲和力的促黄体生成素激素(LH)的适配体,并建立了一个E-AB机器人用于LH的连续监测。E-AB传感器记录适配体特异性结合分子靶标前后构象变化所产生的电化学信号。虽然E-AB传感器能够检测各种目标,包括金属离子、药物分子和蛋白质,但由于其有限的表面积和较大的空间位阻,对生物大分子(>5nm)的检测灵敏度仍处于纳摩尔水平。因此,降低靶标生物大分子在界面上引起的空间位阻以实现灵敏检测一直是研究的重点与难点。
发明内容
本发明的目的在于提供一种超灵敏DNA-生物大分子传感器,构建及其在临床肿瘤样本检测中的应用。解决现有基于界面的检测方法中生物大分子具有较大空间位阻的问题。
本发明构建两相转移的超灵敏DNA-生物大分子传感器,通过将大分子的捕获反应转移到溶液相中,取代下来单链信号探针(~1nm)与界面靠近,大大降低界面的空间位阻,从而提高检测的灵敏度。该传感器具有很好的临床诊断应用前景。
本发明的超灵敏DNA-生物大分子传感器由溶液相、界面相和自组装的金电极组成;其中,溶液相是含有适配体/信号探针(aptamer/signal)双链的反应溶液;自组装的金电极是经辅助探针(helper)自组装的金电极;界面相是溶液相与上述电极之间的界面。所述的靶标生物大分子为激素、蛋白等。
所述的溶液相是加入aptamer/signal双链的反应溶液。所述的aptamer和signal的摩尔浓度比为1:1~1:4,如1:4,1:2,3:4,1:1,3:2,2:1。其中,所述的靶标可以为促黄体生成激素、前列腺特异性抗原等。所述aptamer/signal双链浓度为10nM~50nM。
所述的signal探针为标记了电化学指示剂的DNA链。所述的氧化还原指示剂为亚甲基蓝(MB)或二茂铁(Fc)。所述的界面相是经过辅助探针自组装的金电极与上述溶液相之间的界面。所述金电极直径为0.5~5mm。
所述aptamer和signal杂交缓冲溶液为TM缓冲溶液(50mM MgCl2,20mM Tris,pH8.0)。
所述辅助探针(helper probe,HP)组装浓度为0.1μM~1.5μM。所述PBS缓冲溶液为磷酸盐缓冲液(10mM,pH 7.4),包含NaCl浓度为0.1M~1.5M。
本发明还提供了上述超灵敏DNA-生物大分子传感器的构建方法,具体如下:在检测溶液中加入aptamer/signal双链混合液,当溶液中存在靶标生物大分子时,生物大分子竞争性的与aptamer适配体结合,取代下来signal探针,从而使信号探针进入界面相;信号探针与界面相的辅助探针杂交形成signal/helper双链结构,从而使电化学指示剂靠近电极表面,产生增强的电子传递速率,实现对生物大分子的检测。
其中,所述的aptamer/signal双链制备方法:室温下,aptamer与signal探针按摩尔溶度比为1:1在TM缓冲溶液中反应1小时,杂交形成aptamer/signal双链结构,也可以在85℃下反应10分钟,退火至25℃。
所述的界面相制备方法:将1μL 100μM修饰巯基的辅助探针,10μL 30mM TCEP加入到89μL TM缓冲溶液中,用于还原辅助探针的二硫键。然后滴加3.5-5μL还原后的辅助探针在金电极表面,盖上电极帽孵育过夜。用Milli-Q水冲洗电极,再将电极置于2mM巯基己醇(MCH)溶液中2小时,用以封闭电极表面的空白位点并去除非特异性吸附的寡核苷酸。
其中,金电极的处理方法:分别用0.3mm和0.05mm的氧化铝粉打磨金电极2分钟,然后依次在乙醇和超纯水中超声清洗5分钟。取出后置于0.5M H2SO4溶液中在-0.3~1.5V范围电化学循环伏安扫描清洗3~4次。纯水洗净后将电极转入至0.05M H2SO4中,在-0.2~1.6V范围进行循环伏安扫描,根据所得到的金氧化物溶出峰对应的电荷量确定电极实际表面积,以计算电极平均表面粗糙因子(fr),计算得到的fr=1.35±0.15(n=3)。扫描后用超纯水洗净,氮气吹干备用。
本发明还提供了上述的超灵敏DNA-生物大分子传感器对肿瘤患者血浆样本的检测应用。
本发明构建的超灵敏DNA-生物大分子传感器具有如下特点:
(1)对靶标生物大分子(>5nm)的识别捕获过程在溶液相中进行,即靶标生物大分子竞争性的与双链aptamer/signal中的适配体结合,将信号探针从双链中取代下来,信号探针为ssDNA(~1nm),从而大大减少了界面的空间位阻(图1)。
(2)超灵敏DNA-生物大分子传感器的检测限为10pM,比之前报道的方法低3个数量级。
(3)超灵敏DNA-生物大分子传感器在复杂基质(全血和血浆)中表现出出色的传感性能,检测限均为100pM。
(4)此外,该传感器具有制备简单、成本低廉、易于再生和反复使用的特点,具有应用于即时检测生物大分子的潜力。
附图说明
图1是传感器示意图;其中working electrode为工作电极,aptamer为适配体,signal probe为信号探针,helper probe为辅助探针,macromolecule为靶标生物大分子,solution为反应溶液,interface为反应界面,regeneration为传感器再生,Multichannelpotentiostat为多通道电化学工作站;
图2是PBS缓冲溶液中浓度-电流响应曲线图;横坐标为电压(Potential),纵坐标为电流(Current);
图3是PBS缓冲溶液中检测限考察图;横坐标为标靶生物大分子的浓度(Concentration),纵坐标为电流(Current);
图4是传感器特异性考察图;其中LH为促黄体生成激素,FSH为促卵泡激素,IgG为免疫球蛋白,TSH为促甲状腺激素,SAB为血清白蛋白;
图5是传感器再生和重复使用性能考察图;
图6是电极保存时间考察图;横坐标为保存天数,纵坐标为电流值;
图7是传感器在50%全血中的检测限考察图;a图是50%全血中不同浓度促黄体生成激素下产生的电流信号图;b图是50%全血中空白和100pM促黄体生成激素下的电流信号比较;
图8是传感器在未稀释血浆中的检测限考察图,其中plasma为血浆;a图是血浆中不同浓度促黄体生成激素下产生的电流信号图;b图是血浆中空白和100pM促黄体生成激素下的电流信号比较;
图9是利用传感器检测临床血浆样本中LH浓度图;Healthy为健康人样本,Ovariancancer为卵巢癌患者样本,Breast cancer为乳腺癌患者样本,Prostate cancer为前列腺癌患者样本。
具体实施方式
以下结合具体实施例,对本发明做进一步说明。应理解,以下实施例仅用于说明本发明而非用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
实施例1溶液相反应
针对促黄体生成激素(LH),选择与其特异性结合的适配体。根据适配体,设计与其杂交的信号探针。室温下,将适配体与信号探针以摩尔浓度比为1:1混合在TM缓冲溶液中,杂交形成aptamer/signal双链。当靶标促黄体生成激素存在时,竞争性与适配体结合形成aptamer/LH复合物,并且将信号探针从双链aptamer/signal中释放出来。
表1实施例中涉及的DNA序列
实施例2界面相反应
释放出来的信号探针进入界面相,并且与金电极上的辅助探针杂交,形成signal/helper双链。信号探针与辅助探针杂交形成双链后,指示剂靠近金电极表面,更易于金电极表面发生碰撞,从而加速电子转移,产生电流信号。随着促黄体生成激素浓度的升高,电流信号升高,证明超灵敏DNA-生物大分子传感器是浓度-电流信号响应型(图2)。并且确定该传感器在PBS缓冲溶液中的检测限为10pM(图3)。采用电化学方波伏安法测量电流信号,方波伏安法频率为50Hz。
实施例3传感器性能考察
特异性:当在PBS缓冲溶液中分别加入4种干扰蛋白(促卵泡激素FSH,免疫球蛋白IgG,促甲状腺激素TSH,血清白蛋白SAB)时,电流信号明显低于靶标LH所获得电流信号,表明传感器具有良好的特异性。如图4所示,虽然FSH与LH具有相似的结构,但LH所获得的电流信号约为FSH电流信号的1.5倍。
再生性和重复使用性:采用超纯水冲洗电极60s,去除电极表面的信号探针,就可实现对传感器的再生,如图5所示,传感器再生4次后重新使用的电流值与第一次没有显著型差异,表明传感器具有非常好的再生性和重复使用性能。
保存时间:将电极分别置于25℃和4℃下,分别在第1、7、14和21天测量电流响应。如图6所示,在4℃下,金电极在21天内电流信号基本保持不变,而在25℃下,电流信号随着时间的推移逐渐降低,说明金电极在4℃下至少可以保存21天。
实施例4复杂基质和临床肿瘤样本检测
对于实际复杂基质(如血浆和全血),传感器的检测灵敏度受到质量传输、拥挤效应和环境组分在传感界面上的非特异性吸附等因素的严重影响。因此,我们随后研究了该传感器在实际复杂基质中的可行性。如图7所示,在50%全血中该传感器的检测限为100pM。进一步,考察了该传感器在未稀释血浆中的传感性能。如图8所示,该传感器在未稀释血浆中的检测限为100pM,动力学范围为1nM~1μM。受到以上结果的鼓励,我们利用该传感器检测40例临床血浆样本(健康女性6例,卵巢癌患者4例,乳腺癌患者10例,健康男性10例,前列腺癌患者10例)中LH的浓度,获得了与商品化酶联免疫测定法(ELISA)近乎一致的结果(图9)。
以上所述的,仅为本发明的较佳实施例,并非用以限定本发明的范围,本发明的上述实施例可以做出各种变化。即凡是依据本发明申请的权利要求书及说明书内容所做的简单等效变化与修饰,皆落入本发明的权利要求保护范围。本发明未详尽描述的均为常规技术内容。
序列表
<110> 上海市肿瘤研究所、浙江大学
<120> 一种超灵敏 DNA-生物大分子传感器、构建及其应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 35
<212> DNA
<213> aptamer
<400> 1
tatggtatgc tgtgtggtat ggggtggcgt gctct 35
<210> 2
<211> 15
<212> DNA
<213> signal probe
<400> 2
acacagcata ccata 15
<210> 3
<211> 15
<212> DNA
<213> helper probe
<400> 3
tatggtatgc tgtgt 15
Claims (10)
1.一种超灵敏DNA-生物大分子传感器,其特征在于:所述的DNA-生物大分子传感器由溶液相、界面相和自组装的金电极组成;
所述的溶液相是含有适配体(aptamer)/信号探针(signal)双链的PBS缓冲溶液;
所述的自组装的金电极是经末端修饰巯基的辅助探针(helper)自组装的金电极;
所述的界面相是溶液相与上述电极之间的界面。
2.根据权利要求1所述的一种超灵敏DNA-生物大分子传感器,所述的生物大分子为激素、蛋白。
3.根据权利要求1所述的一种超灵敏DNA-生物大分子传感器,所述的适配体为与靶标生物大分子特异性结合的DNA或RNA。
4.根据权利要求1所述的一种超灵敏DNA-生物大分子传感器,所述的适配体为TATGGTATGCTGTGTGGTATGGGGTGGCGTGCTCT;所述的信号探针为标记了电化学指示剂的DNA链:ACACAGCATACCATA;所述的辅助探针为修饰了巯基的DNA链:TATGGTATGCTGTGT。
5.根据权利要求1所述的平台,其特征在于所述的溶液相中适配体/信号探针的摩尔浓度比为1:1~1:4,所述适配体/信号探针双链浓度为10nM~50nM;所述的辅助探针自组装浓度为0.1μM~1.5μM;所述金电极直径为0.5~5mm。
6.根据权利要求1、2、3、4或5所述的传感器的构建,其特征在于采用如下步骤:适配体预先与信号探针在室温下杂交形成适配体/信号探针双链结构,加入靶标生物大分子(target)后,生物大分子竞争性的与适配体结合形成适配体/靶标生物大分子复合物,信号探针被取代下来并进入界面相;信号探针与金电极上辅助探针杂交形成信号探针/辅助探针双链结构,从而使电化学指示剂靠近电极表面,产生增强的电子传递速率,实现对生物大分子的检测。
7.根据权利要求6所述的超灵敏DNA-生物大分子传感器的构建,其特征在于所述的信号探针标记的电化学指示剂为亚甲基蓝或二茂铁。
8.根据权利要求6所述的超灵敏DNA-生物大分子传感器的构建,其特征在于,所述辅助探针末端修饰巯基,室温下辅助探针通过S-Au键在金电极上自组装形成单分子层。
9.一种如权利要求1所述的超灵敏DNA-生物大分子传感器在生物大分子检测中的应用。
10.如权利要求所述的超灵敏DNA-生物大分子传感器的应用,其特征在于癌症样本中标志物的检测应用。
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