CN110274948A - 一种基于三螺旋分子开关超灵敏测定lps双放大ecl生物传感器及其应用 - Google Patents
一种基于三螺旋分子开关超灵敏测定lps双放大ecl生物传感器及其应用 Download PDFInfo
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Abstract
本发明公开了一种基于三螺旋分子开关超灵敏测定LPS的新型双放大生物传感器及其制法和应用。本发明的技术方案是：通过目标和适配体的特定识别，释放的DNA与多功能分子信标(PMB)杂交，通过Klenow片段和Nt.BbvCI切口酶产生大量片段。将大量片段引入CdTe‑Ru@SiO2纳米球/TPrA‑ECL‑三螺旋传感系统，打开三螺旋结构，通过氯化血红素有效淬灭ECL产生响应，实现三螺旋分子“开‑关”信号响应模式检测目标LPS。
Description
技术领域：
本发明涉及一种用于多功能电化学发光(ECL)“关闭”检测双放大过程脂多糖(LPS)的生物传感器；本发明还涉及所述生物传感器的制备方法及其检测LPS的分析应用。
背景技术：
脱氧核糖核酸技术[a)C.-H.Lu,B.Willner,I.Willner,ACS Nano 2013,7,8320–8332；b)O.I.Wilner,I.Willner,Chem.Rev.2012,112,2528–2556；c)C.Teller,I.Willner,Curr.Opin.Biotechnol.2010,21,376–391；d)N.C.Seeman,Nature 2003,421,427–431；e)D.Y.Zhang,G.Seelig,Nat.Chem.2011,3,103–113.]是一个快速发展的研究领域。在核酸的基本序列中编码的信息已被广泛用于构建一维、二维和三维结构，设计DNA开关[F.Wang,X.Liu,I.Willner,Angew.Chem.Int.Ed.2015,54,1098–1129；Angew.Chem.2015,127,1112–1144.]和机器，使用核酸作为逻辑门操作的材料。
电致化学发光(ECL)作为一种强大的分析技术，由于其具有高灵敏度、低背景、操作简单和优异的可控性等优点而受到越来越多的关注[Liu Y,Lei J,Huang Y,etal.Analytical Chemistry,2014,86(17):8735–8741；Ma W,Xu L,de MouraA F,etal.Chemical Reviews,2017,117(12):8041–8093.]，并且被应用于多种生物标志物检测中。与此同时，许多具有较强发光性能和良好的电化学稳定性的ECL发光材料也不断被开并被应用于生物传感之中，例如鲁米诺[Xu L,Sun M,MaW,et al.Materials Today,2016,19:595–606.]、量子点[Rohrbach F,F,Fichte MAH,et al.Angewandte ChemieInternational Edition,2013,52(45):11912-11915.]、钌及其衍生物[Deng C,Chen J,Nie L,et al.Analytical Chemistry,2009,81(24):9972-9978.]等。特别是量子点材料，因其较好的生物相容性备受青睐[Rohrbach F,F,Fichte MAH,et al.AngewandteChemie International Edition,2013,52(45):11912-11915.]。
三重核酸作为用于开发DNA纳米结构和材料的丰富"工具箱"的一部分而引起了人们的兴趣。人们利用三重DNA不仅仅是一种研究识别元件,也作为一个功能结构开关单元,它允许在目标识别时生成输出信号。用于进一步开发DNA纳米技术和基于核酸的功能材料。这些初步结果凸显了DNA三叉戟在未来纳米医学应用中的意义。除了对三重DNA结构的基本兴趣外,生物这些系统的影[R.Zain,J.S.Sun,Cell.Mol.Life Sci.2003,60,862–870.]及其潜在的治疗应用。
本发明设计了提出了一种基于三螺旋分子开关超灵敏测定LPS的新型双放大生物传感平台。三种DNA通过目标和适配体的特定识别，磁性释放分离。释放的DNA通过几何级数与多功能分子信标(PMB)杂交，通过Klenow片段和Nt.BbvCI切口酶产生大量片段。将大量片段引入三螺旋传感系统，从而打开三螺旋结构并产生ECL响应。
发明内容：
本发明的目的是提供基于三螺旋分子开关超灵敏的新型双放大生物传感平台测定LPS。具体包括以下步骤：
CdTe-Ru@SiO2纳米球的制备：采用两步法制备水溶性CdTe QDs。一：在氮气环境中将50.0mgNaBH4和80.0mg Te粉末在45℃下加入3.0mL超纯水中30分钟，得到透明的深紫色NaHTe溶液。将2.5mmol的CdCl2溶解于63mL的超纯水，加入55μLMPA，氮气脱气，用0.2mol L- 1NaOH调pH至9.0。将250μLNaHTe溶液快速注入CdCl2反应烧瓶中，加热至130℃并在氮气环境下回流12小时。冷却至室温后，得到橙红色CdTe QDs溶液。二：制备CdTe-Ru@SiO2纳米球。制备的CdTe QD(200μL)与[Ru(bpy)3]2+(80mM,170μL)在锥形瓶中反应过夜。恒定搅拌下将环己烷，TX-100和正己醇注入混合物中25分钟。快速注入前体TEOS(100μL)，60μLNH3·H2O引发聚合。密封避光反应24h，丙酮离心(12000rpm,10min)，乙醇纯化。将获得的CdTe-Ru@SiO2纳米球分散PBS溶液中。
适配器改性磁珠的制备：先分离100μL羧基修饰的磁珠(粒径：2.0～3.0μm；量：10mL；1％(w/v)。用0.5mLPBS缓冲液(pH 7.4，0.1MNa2HPO4·12H2O，0.1MNaH2PO4·2H2O，0.1MKCl)洗涤三次。将磁珠在含有10mgNHS和20mg EDC的200μL 0.1M PBS缓冲液中活化1小时，同时在室温下轻轻摇动，弃去上清液。之后，将100μL 1.0μM氨基修饰的LPS适体加入到活化的磁珠溶液中，所得混合物在恒温(37℃)振荡器中反应6小时。磁力分离并用200μLPBS缓冲液(0.1M，pH 7.4)洗涤三次后，在37℃下将适体-磁珠分散在200μL0.1M PBS缓冲液(0.1M，pH 7.4)中1小时。在37℃下将300μL的三个mDNA(10-5M每个)加入上述溶液中2小时。用PBS缓冲液(0.1M，pH 7.4)洗涤三次后，将杂交复合物重悬于200μLPBS缓冲液(0.1M，pH 7.4)中以备进一步使用。将最终的适体-mDNA-磁珠溶液储存在4℃的冰箱中。
目标循环扩增反应：在10μL的悬浮液中加入一定浓度的LPS(15μL)。然后在37℃孵育混合物2小时,释放m1，m2，m3。磁分离后，m1、m2和m3被留在上清液中。将2.5μL 10×NE缓冲液(500mMNaCl,100mM Tris-HCl,100mM MgCl2，10mM dithiothreitol(DTT),pH 7.9at25℃)，0.5μL 0.1μM的PMB，0.6μL 5U/μL片段(3′-5′exo-)，0.6μL 10U/μL Nt.BbvCInickase，1μL的10mM核苷酸，20μL上层清液都先后加入小管。通过在80℃下加热灭活20min终止反应，将所得溶液冷却至室温。此时，该溶液含有大量被替换的带切口的片段(NFs)。
三重螺旋DNA的构建：45μL孵化缓冲液(10mM PB,20mM NaCl,2.5mM MgCl2,pH5.2)中加入了DNA S1(50μL,1.0μM)和DNA S2(50μL,1.0μM)，室温下孵育2小时,形成三螺旋DNA。
LPS的ECL检测：分别用1.0，0.3和0.05μm氧化铝粉末抛光金电极，超纯水冲洗。将电极置于50％甲醇溶液中超声处理3min。接着，电极在0.5M H2SO4中进行电化学清洗，电位扫描从-0.3V到1.5V，直到得到可重复的循环伏安图，然后超纯水冲洗，氮气干燥。将10μLCdTe-Ru@SiO2纳米球溶液滴于裸露的金电极表面，在空气中干燥。将电极浸入EDC/NHS(20mg mL-1/10mg mL-1)溶液中20min，活化羧基，超纯水冲洗。然后将电极浸泡在三螺旋DNA溶液中6小时，超纯水冲洗。将电极浸泡在NFs中2小时，超纯水冲洗。最后，将10μL氯化血红素(7*10-4M)移液到修饰电极上30分钟，以形成氯化血红素/G-四链体纳米结构。用超纯水冲洗改性电极，并在空气中干燥。ECL检测在含10mM Tpr的PBS缓冲液(0.1M,pH＝7.4)中进行。扫描电位为0.2～1.25V，扫描速率为0.1V/s，光电倍增管为-500V。
本发明主要优点在于：首先，构建了一种新型的CdTe-Ru@SiO2纳米球/TPrAECL平台，通过氯化血红素有效淬灭，实现三螺旋分子开关“开-关”信号响应模式。其次，在LPS的超灵敏检测过程中首次提出了双放大过程。此外，与传统的生物检测方法相比，开发的生物传感平台具有优异的稳定性，灵敏度和选择性，宽线性范围以及低检测限。值得指出的是，双信号平台在生物分析、早期临床诊断、生物医学研究等方面具有广阔的应用前景。
附图说明：
图1(A)适配器与LPS结合并释放m1、m2、m3；(B)循环扩增过程,其中m1、m2、m3和PMB分子由几何组合进展；(C)基于三重螺旋分子开关的ECL“关闭”检测LPS。
图2在含有不同物质的PBS中电极检测的ECL强度-时间曲线：(a)CdTe-Ru@SiO2纳米球+10mM TPrA，(b)CdTe-Ru@SiO2纳米球+10mMTPrA+0.7mM氯化血红素。
图3双扩增策略的PAGE分析：(A)LPS-适体结合诱导m1，m2，m3，(B)循环扩增过程的DNA序列释放过程。
图4生物传感器对应不同浓度目标LPS的ECL信号响应(fg/mL)：(a)0；(b)0.1；(c)1.0；(d)10；(e)100；(f)1000；(g)10000；(h)100000(从a到h)。(B)ΔECL(扣除背景，ΔIECL＝I0-I)和LPS浓度(插图：检测LPS的对数矫正图)之间的关系，误差棒代表三个平行实验的标准偏差。
图5(A)ECL生物传感器检测LPS(1.0pg/mL)的特异性与CEA，HL，TB，AFP(1.0pg/mL)，空白(无LPS)和含有1.0pg的混合物的信号比较。
具体实施方式：
实施例1双放大生物传感平台的制备及对LPS的检测
目标循环扩增反应：在适配器改性磁珠制备中取得的悬浮液取10μL，加入一定浓度的LPS(15μL)。然后在37℃孵育混合物2小时，释放m1，m2，m3。磁分离后，m1、m2和m3被留在上清液中。2.5μL 10×NE缓冲液(500mMNaCl，100mM Tris-HCl，100mM MgCl2,10mMdithiothreitol(DTT)，pH 7.9at 25℃)，0.5μL 0.1μM的PMB，0.6μL 5U/μL片段(3′-5′exo-)，0.6μL 10U/μLNt.BbvCI nickase，1μL的10mM核苷酸，20μL上层清液都先后加入小管。通过在80℃下加热灭活20分钟终止反应，将所得溶液冷却至室温。此时，该溶液含有大量被替换的带切口的片段(NFs)。
三重螺旋DNA的构建：在45μL孵化缓冲液(10mM PB，20mMNaCl,2.5mM MgCl2，pH5.2)中加入了DNA S1(50μL,1.0μM)和DNA S2(50μL,1.0μM)，室温下孵育2小时,形成三螺旋DNA。
LPS的ECL检测：分别1.0，0.3和0.05μm氧化铝粉末抛光金电极，超纯水冲洗。将电极置于50％甲醇溶液中超声处理3min。接着，电极在0.5M H2SO4中进行电化学清洗，电位扫描从-0.3V到1.5V，直到得到可重复的循环伏安图，然后超纯水冲洗，氮气干燥。将10μLCdTe-Ru@SiO2纳米球溶液滴于裸露的金电极表面，在空气中干燥。将电极浸入EDC/NHS溶液中活化20min。然后将电极浸泡在三螺旋DNA溶液中6小时。将电极浸泡在NFs中2小时，超纯水冲洗。最后，将10μL氯化血红素(7*10-4M)移液到修饰电极上3分钟。ECL检测在含10mMTPrAPBS缓冲液中进行。
实施例2双放大生物传感平台的制备及对LPS的检测
将“2.5μL 10×NE缓冲液(500mM NaCl，100mM Tris-HCl，100mM MgCl2,10mMdithiothreitol(DTT)，pH 7.9at 25℃)，0.5μL0.1μM的PMB，0.6μL 5U/μL片段(3′-5′exo-)，0.6μL 10U/μLNt.BbvCI nickase，1μL的10mM核苷酸，20μL上层清液都先后加入小管。”改为“2.5μL 10×NE缓冲液(500mM NaCl，100mM Tris-HCl，100mM MgCl2,10mMdithiothreitol(DTT)，pH 7.9at 25℃)，0.5μL0.1μM的PMB，0.6μL 7U/μL片段(3′-5′exo-)，0.6μL 10U/μLNt.BbvCI nickase，1μL的10mM核苷酸，20μL上层清液都先后加入小管。”制备的其他条件同实施例1，得到形貌与性质类似于实施例1的生物传感平台。对LPS检测的结果同实施例1。
实施例3双放大生物传感平台的制备及对LPS的检测
将“2.5μL 10×NE缓冲液(500mM NaCl，100mM Tris-HCl，100mM MgCl2,10mMdithiothreitol(DTT)，pH 7.9at 25℃)，0.5μL0.1μM的PMB，0.6μL 5U/μL片段(3′-5′exo-)，0.6μL 10U/μLNt.BbvCI nickase，1μL的10mM核苷酸，20μL上层清液都先后加入小管。”改为“2.5μL 10×NE缓冲液(500mM NaCl，100mM Tris-HCl，100mM MgCl2,10mMdithiothreitol(DTT)，pH 7.9at 25℃)，0.5μL0.1μM的PMB，0.6μL 5U/μL片段(3′-5′exo-)，0.6μL 12U/μLNt.BbvCI nickase，1μL的10mM核苷酸，20μL上层清液都先后加入小管。”制备的其他条件同实施例1，得到形貌与性质类似于实施例1的生物传感平台。对LPS检测的结果同实施例1。
实施例4双放大生物传感平台的制备及对LPS的检测
将“然后将电极浸泡在三螺旋DNA溶液中6小时，超纯水冲洗。”改为“然后将电极浸泡在三螺旋DNA溶液中8小时，超纯水冲洗。”制备的其他条件同实施例1，得到形貌与性质类似于实施例1的生物传感平台。对LPS检测的结果同实施例1。
实施例5双放大生物传感平台的制备及对LPS的检测
将“将电极浸泡在NFs中2小时，超纯水冲洗。最后，10μL氯化血红素(7*10-4M)移液到修饰电极上30分钟，以形成氯化血红素/G-四链体纳米结构。”改为“将电极浸泡在NFs中2小时，超纯水冲洗。最后，将10μL氯化血红素(7*10-4M)移液到修饰电极上40分钟，以形成氯化血红素/G-四链体纳米结构。”制备的其他条件同实施例1，得到形貌与性质类似于实施例1的生物传感平台。对LPS检测的结果同实施例1。
Claims (3)
1.一种基于三螺旋分子开关超灵敏测定LPS的新型双放大生物传感器及其制法和应用。其特征是：通过目标和适配体的特定识别，磁性分离释放的DNA与多功能分子信标(PMB)杂交,利用聚合酶和内切酶放大反应过程产生大量的DNA片段。该片段引入三螺旋传感系统,打开三螺旋结构并利用氯化血红素淬灭ECL产生响应，实现对目标的检测。该多功能生物传感器具有良好的分析性能，较宽的线性范围和较低的检测极限。
2.一种制备权利要求1所述的基于三螺旋分子开关超灵敏测定LPS的新型双放大生物传感器的方法和应用，其特征方法由下列步骤组成：
步骤1.CdTe-Ru@SiO2纳米球的制备
采用两步法制备水溶性CdTe QDs。根据Gu等人公布的方法制备碲化氢钠(NaHTe)。在氮气环境中，将50.0mg NaBH4和80.0mgTe粉末在45℃下加入3.0mL超纯水中30分钟。在Te粉末完全消失后，此时溶液变为深紫色，得到透明的NaHTe溶液。将2.5mmol的CdCl2溶解于63mL的超纯水中，然后加入5μL的MPA，用氮气脱气，用0.2mol L-1NaOH将pH调节至9.0。然后将250μL NaHTe溶液快速注入CdCl2反应烧瓶中，加热至130℃并在氮气环境下回流12小时。冷却至室温后，得到橙红色CdTe QDs溶液。
制备的CdTe QD(200μL)与[Ru(bpy)3]2+(80mM,170μL)在锥形瓶中反应过夜。然后环己烷，TX-100在恒定搅拌下和正己醇注入混合物中25分钟。快速注入前体TEOS(100μL)后，加入60μLNH3·H2O以引发聚合反应。在密封容器中避光反应24小时后，用丙酮分离所得产物，离心(12000rpm，10分钟)用乙醇纯化。将获得的CdTe-Ru@SiO2纳米球分散在PBS溶液中。
步骤2.适配器改性磁珠的制备
首先分离100μL羧基修饰的磁珠(粒径：2.0～3.0μm；量：10mL，1％(w/v))用0.5mL PBS缓冲液(pH7.4,0.1M Na2HPO4·12H2O,0.1M NaH2PO4·2H2O,0.1M KCl)洗涤三次。然后将磁珠在含有10mg NHS和20mg EDC的200μL的0.1M PBS缓冲液中活化1小时，同时在室温下轻轻摇动，弃去上清液。将100μL 1.0μM氨基修饰的LPS适体加入到活化的磁珠溶液中，并使得到的混合物在37℃下在恒温振荡器中反应6小时。磁力分离并用200μL PBS缓冲液(0.1M，pH7.4)洗涤三次后，37℃下将适体-磁珠分散在200μL 0.1M PBS缓冲液(0.1M，pH 7.4)中1小时。接着，将300μL的三种mDNA(每个10-5M)在37℃下加入上述溶液中2小时。用PBS缓冲液(0.1M，pH 7.4)洗涤三次后，将杂交复合物重悬于200μL PBS缓冲液(0.1M，pH 7.4)中以备进一步使用。将最终的适体-mDNA-磁珠溶液储存在4℃的冰箱中。
步骤3目标循环扩增反应
在10μL的悬浮液中加入一定浓度的15μL LPS。然后,在37℃孵育2小时,释放m1,m2,m3。磁分离后,m1、m2和m3被留在上清液中。然后2.5μL 10×NE缓冲液2(氯化钠500mM,100mMTris-HCl,100mM MgCl2,10mM二硫苏糖醇(DTT)，pH值7.925℃),0.5μL μM PMB的0.1，0.6μL5U/μL大片段(3′－5′exo-)，0.6μL 10U/μL Nt.BbvCI nickase,1μL的10mM核苷酸，20μL上层清液都先后加入小管。轻轻混合后,在37℃孵育90分钟。随后,反应在80℃下热失活，终止20分钟,溶液冷却到室温。产生大量的NFs片段。
步骤4三重螺旋DNA的构建
在45μL孵化器中加入了DNA S1(50μL,1.0μM)和DNA S2(50μL,1.0μM)(10mM PB,20mMNaCl,2.5mM MgCl2,pH 5.2),并在室温下孵育2小时,形成三螺旋DNA。
3.根据权利要求2所述的LPS检测方法，其特征是：分别用1.0、0.3、0.05μm氧化铝粉末抛光金电极，用超纯水冲洗。将电极置于50％甲醇溶液中超声处理3min；接下来，电极在0.5M H2SO4中进行电化学清洗，电位扫描从-0.3V到1.5V，直到得到可重复的循环伏安图，超纯水冲洗，氮气干燥。随后将10μL CdTe-Ru@SiO2纳米球溶液滴于裸露的金电极表面，在空气中干燥。然后将电极浸入EDC/NHS(20mg·mL-1/10mg·mL-1)溶液中20min活化羧基，用超纯水冲洗。将电极浸泡在三螺旋DNA溶液中6小时，超纯水冲洗。将电极浸泡在NFs中2小时，超纯水冲洗。最后,10μL氯高铁血红素(7*10-4M)是用移液器吸取到30分钟的修饰电极，形成氯化铁血红素/G-四链体纳米结构。用超纯水冲洗改性电极，并在空气中干燥。ECL检测在含10mm TPrA的PBS缓冲液(0.1M,pH＝7.4)中进行。扫描电位为0.2～1.25V，扫描速率为0.1V/s，光电倍增管为-500V。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111157598A (zh) * | 2020-01-04 | 2020-05-15 | 青岛科技大学 | 一种dna纳米线放大的共敏光电检测平台及其制法和应用 |
| WO2022169890A1 (en) | 2021-02-04 | 2022-08-11 | Fresenius Medical Care Holdings, Inc. | Lipopolysaccharide (lps) aptamers and associated methods |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110014293A1 (en) * | 2009-07-14 | 2011-01-20 | Dar-Bin Shieh | Cleavage kit, and gene therapy by using the same and nucleic acid cleavage detection apparatus |
| CN102507689A (zh) * | 2011-10-19 | 2012-06-20 | 青岛科技大学 | 一种检测凝血酶的电化学发光传感器的制备方法及应用 |
| CN105510420A (zh) * | 2015-12-20 | 2016-04-20 | 青岛科技大学 | 一种基于磁珠分离和dna标记金纳米粒子探针检测atp含量的方法 |
| WO2018049170A1 (en) * | 2016-09-09 | 2018-03-15 | Board Of Regents, The University Of Texas System | Sensors and methods for making and using the same |
| CN109211997A (zh) * | 2018-09-14 | 2019-01-15 | 东南大学 | 一种检测β-淀粉样蛋白的基于THMS的电化学发光适体传感器及其制备方法和应用 |
| CN109444117A (zh) * | 2018-11-05 | 2019-03-08 | 青岛科技大学 | 一种基于银纳米簇猝灭效应和多重放大技术的电化学发光生物传感器及其应用 |
| CN109959691A (zh) * | 2019-04-15 | 2019-07-02 | 济南大学 | 一种基于级联光电活性材料和三螺旋分子开关检测核酸的方法 |
-
2019
- 2019-07-11 CN CN201910624183.7A patent/CN110274948B/zh active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110014293A1 (en) * | 2009-07-14 | 2011-01-20 | Dar-Bin Shieh | Cleavage kit, and gene therapy by using the same and nucleic acid cleavage detection apparatus |
| CN102507689A (zh) * | 2011-10-19 | 2012-06-20 | 青岛科技大学 | 一种检测凝血酶的电化学发光传感器的制备方法及应用 |
| CN105510420A (zh) * | 2015-12-20 | 2016-04-20 | 青岛科技大学 | 一种基于磁珠分离和dna标记金纳米粒子探针检测atp含量的方法 |
| WO2018049170A1 (en) * | 2016-09-09 | 2018-03-15 | Board Of Regents, The University Of Texas System | Sensors and methods for making and using the same |
| CN109211997A (zh) * | 2018-09-14 | 2019-01-15 | 东南大学 | 一种检测β-淀粉样蛋白的基于THMS的电化学发光适体传感器及其制备方法和应用 |
| CN109444117A (zh) * | 2018-11-05 | 2019-03-08 | 青岛科技大学 | 一种基于银纳米簇猝灭效应和多重放大技术的电化学发光生物传感器及其应用 |
| CN109959691A (zh) * | 2019-04-15 | 2019-07-02 | 济南大学 | 一种基于级联光电活性材料和三螺旋分子开关检测核酸的方法 |
Non-Patent Citations (3)
| Title |
|---|
| GUIFEN JIE 等: "Magnetic Electrochemiluminescent Fe3O4/CdSe–CdS Nanoparticle/Polyelectrolyte Nanocomposite for Highly Efficient Immunosensing of a Cancer Biomarker", 《CHEM. EUR. J.》 * |
| TAO YU 等: "Highly Sensitive Colorimetric Cancer Cell Detection Based on Dual Signal Amplification", 《ACS APPL. MATER. INTERFACES》 * |
| 杨云旭 等: "三螺旋DNA电致化学发光生物传感器的组装与表征", 《青岛科技大学学报（自然科学版）》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111157598A (zh) * | 2020-01-04 | 2020-05-15 | 青岛科技大学 | 一种dna纳米线放大的共敏光电检测平台及其制法和应用 |
| WO2022169890A1 (en) | 2021-02-04 | 2022-08-11 | Fresenius Medical Care Holdings, Inc. | Lipopolysaccharide (lps) aptamers and associated methods |
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