CN110592191A - 一种基于酶催化循环及二硫化钼吸附介导可视化检测核酸的方法 - Google Patents

一种基于酶催化循环及二硫化钼吸附介导可视化检测核酸的方法 Download PDF

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CN110592191A
CN110592191A CN201910882957.6A CN201910882957A CN110592191A CN 110592191 A CN110592191 A CN 110592191A CN 201910882957 A CN201910882957 A CN 201910882957A CN 110592191 A CN110592191 A CN 110592191A
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nucleic acid
molybdenum disulfide
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CN110592191B (zh
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汪联辉
朱煜
朱丹
赵东霞
晁洁
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Nanjing Post and Telecommunication University
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Abstract

本发明公开一种基于酶催化循环及二硫化钼介导吸附可视化检测核酸的方法，包括：将巯基或polyA修饰的捕获序列组装在金纳米粒子表面，构建纳米金捕获探针；向含有目标核酸的待测体系中加入捕获探针，目标核酸与捕获探针完成杂交后进行循环剪切反应；向完成剪切反应体系中加入二硫化钼纳米片，静置反应；取步骤反应后体系的上清液，肉眼观察上清液颜色的变化，并通过紫外分光光度计测量上清液的吸光值变化。本发明基于目标核酸触发的酶循环反应，利用二硫化钼与纳米金探针的吸附进行信号输出，通过裸眼观察或比色光谱即可实现对目标核酸的现场原位快速检测；本发明操作简单、反应条件温和、灵敏度高，选择性好，具有广阔的应用前景。

Description

一种基于酶催化循环及二硫化钼吸附介导可视化检测核酸的 方法
技术领域
本发明属于比色试剂研究及核酸检测领域，具体涉及一种基于酶催化循环及二硫化钼吸附介导可视化检测核酸的方法。
背景技术
在过去的几年中，核酸检测在医学研究和诊断以及食品和药物工业监测方面具有重要的应用，受到了高度关注。聚合酶链反应(PCR)是当前核酸检测的经典技术。然而，PCR需要针对不同的靶标设计复杂的引物，步骤繁琐，成本高昂，无法满足临床检测的需求。纳米生物传感技术的发展为核酸检测提供了新的契机，通常通过在纳米传感体系中引入与目标核酸相互补的捕获序列用于核酸的识别和检测。其中，基于捕获序列和核酸靶标分子1:1杂交作用机制的分析方法具有有限的灵敏度和检测能力，已无法满足当前生物分析中高灵敏度和低检测限的需求。许多等温扩增技术和酶扩增循环技术，包括滚环扩增(RCA)、链置换扩增(SDA)、解旋酶扩增(HAD)和杂交链式反应(HCR)，都正革新着核酸检测的里程(Lee,S.H.,Park,S.M.,et al.,Biosens.Bioelectron.,2019,141,111448；Reid,M.S.,Chris,L.X.,et al.,Angew.Chem.Int.Ed.,2018,37,11856-11866)。其中，酶催化循环反应可以通过酶特异性地剪切核酸分子，有效地提高靶标利用率，扩增检测信号。此外，酶兼具高效性和专一性，在提高生物检测灵敏度的同时，也提高了生物检测的特异性(Qian,C.,Wang,R.,et al.,Anal.Chem.Acta,2019,1050,1-15；Xie.,X.,Xu,W.,Acc.Chem.Res.,2012,9,1511-1520)。例如，核酸外切酶III是一类作用于双链DNA的核酸剪切酶，可沿平末端或3’凹陷末端从3’→5’方向逐步催化去除单链单核苷酸，而不影响另一条互补序列的完整性(Angew.Chem.Int.Ed.,2017,56,1855-1858)。
比色检测作为一种现场诊断方法，具有操作简单、实时便携、现场分析等优点，一直是化学分析和医学检测中的研究热点。比色检测通过传感体系中有色试剂或纳米材料的颜色变化，可以直观地诊断出目标分子的存在与否(Tang,L.,Li,J.ACS Sensors,2017,2,857-875)。但现有比色检测存在灵敏度不高、体系抗干扰性差等缺陷(Zhang,Y.,McKelvie,L.,et al.,Talanta,2016,152,410-422)。常用的显色纳米材料金纳米粒子由于具备独特的光学特性、强大的表面等离子共振吸收和高消光系数，已成为便携式仪器甚至肉眼监测分析目标的理想材料。发展操作简单、具有高灵敏度和选择性的比色检测方法对核酸检测领域至关重要。
发明内容
发明目的：针对现有技术存在可视化核酸检测中灵敏度低、操作复杂的等问题，本发明提供了一种基于酶催化循环及二硫化钼吸附介导可视化检测核酸的方法。本方法将可识别目标核酸的单链捕获序列通过巯基修饰或尾部延伸的polyA修饰在金纳米粒子表面构建纳米金捕获探针，随后加入目标核酸杂交形成双链结构，用核酸外切酶III特异性切割后，加入二硫化钼纳米片进行显色分析。若体系中不存在目标核酸，捕获序列以单链形式存在，核酸外切酶III与单链捕获序列之间无特异反应，捕获序列的单链粘性末端可通过范德华力与二硫化钼吸附，使二硫化钼层层堆叠，造成纳米金和二硫化钼的聚沉；当存在目标核酸时，目标核酸通过碱基互补配对原则与金纳米粒子表面的捕获序列形成远端平齐的双链DNA，捕获序列可被核酸外切酶III从3’→5’方向逐步剪切，并释放出目标核酸被下一条捕获序列捕获，继续引发循环的酶切反应。因此，少量的目标核酸便可使纳米金表面的捕获序列几乎被核酸外切酶III完全剪切，不引起二硫化钼和纳米金的聚沉，在溶液中保持分散状态。该现象可通过肉眼观察或紫外可见分光光度计定量检测，实现对目标核酸的超灵敏快速检测。
技术方案：为了实现上述目的，如本发明所述一种基于酶催化循环及二硫化钼介导吸附可视化检测核酸的方法，包括如下步骤：
(1)将巯基或polyA修饰的捕获序列组装在金纳米粒子表面，构建纳米金捕获探针；所述的捕获序列端部修饰有巯基基团或polyA基团，捕获序列为和目标核酸互补的序列，通过加盐老化法将捕获序列组装在金纳米粒子表面；
(2)向含有目标核酸的待测体系中加入捕获探针，目标核酸与捕获探针完成杂交后向体系添加含有核酸外切酶III的反应液，进行循环剪切反应；
(3)向步骤(2)的完成剪切反应体系中加入二硫化钼纳米片，静置反应；
(4)取步骤(3)反应后体系的上清液，肉眼观察上清液颜色的变化，并通过紫外分光光度计测量上清液的吸光值变化。
其中，所述捕获序列通过加盐老化组装在金纳米粒子表面。
作为优选，所述金纳米粒子的粒径为10-50nm。更优选金纳米粒子为15nm。
其中，所述加盐老化为将polyA或巯基修饰的目标核酸捕获序列与金纳米粒子按一定比例混合，在室温下轻摇过夜；分批多次加入1M PBS(1M NaCl，100mM PB，pH＝7.4)溶液，继续室温下轻摇过夜；离心去除多余的捕获序列，离心清洗，得到的捕获探针分散在0.1M PBS(0.1M NaCl，10mM PB，pH＝7.4)溶液中储存。
具体为：将polyA或巯基修饰的目标核酸捕获序列与金纳米粒子以200：1的摩尔比例混合，在25℃室温下轻摇过夜；向溶液中按体积比例加入1M PBS(1M NaCl，100mM PB，pH＝7.4),每隔30min加一次，分五次加入，使得混合溶液终浓度为0.1M PBS(0.1M NaCl，10mMPB，pH＝7.4)，继续在25℃室温下轻摇过夜；离心(12000rpm，20min，15℃)去除多余的捕获序列，并用0.1M PBS(pH＝7.4)溶液反复离心清洗三次。得到的捕获探针分散在储备溶液(0.1M NaCl，10mM PB，pH＝7.4)中。
其中，所述捕获序列为与待检测目标核酸互补的序列。
其中，所述步骤(2)中的反应液或者杂交缓冲液优选为pH＝7.4的PBS溶液(100mMNaCl，10mM PB，2mM MgCl2)。即进行杂交反应和酶切反应时的溶液。
其中，步骤(2)所述目标核酸与捕获探针的杂交时间为10-30min。作为优选，所述目标核酸与捕获探针的杂交时间为10min。
进一步地，步骤(2)所述循环剪切反应为采用核酸外切酶III进行，所述核酸外切酶III的终浓度为0.05-0.2U/μL，剪切时间为20-90min。作为优选，所述核酸外切酶III的终浓度为0.1U/μL，剪切时间为30min。
作为优选，步骤(3)所述二硫化钼纳米片的尺寸为100-300nm*100-300nm，终浓度优选为5-18μg/mL。更优选的为二硫化钼纳米片的尺寸为200nm*200nm。
其中，步骤(3)所述静置反应时间为10-90min。更优选静置70min。
本发明可视化检测核酸的方法中使用二硫化钼(MoS2)，是一种典型的二维过渡金属硫化物(Transition Metal Dichalcogenides,TMDs)，具有和石墨烯类似的二维层状结构，水溶性比石墨烯更强，生物毒性更低，适合于生物环境下的检测分析。此外，二硫化钼出色的荧光淬灭效应、宽广的比表面积有利于使它成为具有优势的传感平台。而且，二硫化钼可以通过范德华力与单链核酸之间形成强吸附，由此可以通过改变核酸的存在状态调节核酸和二硫化钼之间的吸附力。二硫化钼溶于水后呈黑色分散状态溶液，若在溶液中发生聚集，则沉聚成黑色颗粒沉在底部，用肉眼可明显观察到颜色和状态变化。本发明结金纳米粒子和二硫化钼的优异特性，发明了一种基于酶催化循环反应及二硫化钼介导可视化检测核酸的方法，成功实现了对核酸分子的快速可视化检测。
本发明将核酸外切酶III与单链捕获探针修饰的纳米金作用，通过目标核酸的识别实现循环切割反应，利用二硫化钼与单链DNA的亲和力完成二硫化钼介导的纳米颗粒沉聚，通过肉眼或紫外可见分光光度计定量检测目标核酸。制备方法：通过巯基修饰基团或尾部延伸的polyA将可识别目标核酸的单链捕获序列修饰在金纳米粒子表面；加入待测样品并用核酸外切酶III处理；加入二硫化钼纳米片进行显色分析。
有益效果：与现有技术相比，本发明具有如下优点：
(1)本发明利用基于目标核酸触发的酶循环反应，可以有效提高检测灵敏度，选择性好；
(2)本发明创新性地利用二硫化钼与纳米金探针的吸附进行信号输出，通过裸眼观察或比色光谱即可实现对目标核酸的现场原位快速检测；
(3)本发明操作简单，反应条件温和，成本低廉，在医学诊断和便携式检测中具有广阔的应用前景；
(4)通过改变polyA长度，polyA修饰的捕获序列可以在纳米金表面实现捕获序列组装密度和构型的可控调节，有利于界面上核酸杂交反应的进行。
附图说明
图1为本发明制备的基于酶催化循环及二硫化钼介导吸附可视化检测核酸的机理示意图；
图2为本发明制备的纳米探针中二硫化钼的透射电镜图片；
图3为本发明制备的金纳米粒子和纳米金捕获探针的紫外-可见吸收光谱图；
图4为本发明制备的纳米探针溶液与二硫化钼纳米片的孵育时间优化；
图5为本发明制备的纳米探针溶液与二硫化钼纳米片的孵育浓度优化；
图6为本发明制备的可视化探针随目标核酸浓度变化的颜色变化及其对应的紫外-可见吸收光谱图；
图7为本发明制备的可视化探针对目标核酸检测的特异性实验；
图8为本发明制备的可视化探针对目标核酸、碱基错配核酸响应的颜色变化及其对应的紫外-可见吸收光谱图。
具体实施方式
以下结合实施例和附图对本发明作进一步说明。
本发明是以加盐老化或快速组装法将巯基或polyA修饰的捕获序列组装在金纳米粒子表面，构建纳米金捕获探针。捕获序列通过Au-S化学键或polyA与金的共价吸附连接在纳米金表面。利用核酸互补配对原理使纳米金上的捕获序列和目标序列DNA互补杂交形成双链结构，加入核酸外切酶III对双链结构中的捕获序列进行特异性切割，释放出目标序列参与循环切割反应。反应完成后，加入二硫化钼纳米片进行吸附显色。显色完成后，取体系的上清液，肉眼观察上清液颜色的变化，或通过紫外分光光度计测量上清液的吸光值变化，其原来示意图如图1所示。
本发明实施例中所有原料和试剂如无特殊说明，均市售可得。
实施例1
纳米金-捕获探针的制备：
(1)将polyA修饰的目标核酸捕获序列与15nm金纳米粒子以200：1的摩尔比例(如终浓度600nM DNA：3nM纳米金)混合，在25℃室温下轻摇过夜，捕获序列为：SEQ ID NO.15’-AAAAAAAAAAAAAAAAAAAACTCTCTAAAATCACT-3’(polyA修饰的捕获序列)；上述步骤中也可以采用巯基修饰的目标核酸捕获序列，SEQ ID NO.2：5’-SH-CTCTCTAAAATCACT-3’(巯基修饰的捕获序列)，后续效果与polyA修饰的目标核酸捕获序列一致。
(2)向步骤(1)的溶液中按体积比例加入1M PBS(100mM PB，1M NaCl，pH＝7.4)，每隔30min加一次，分五次加入，使得混合溶液终浓度为0.1M PBS(10mM PB，0.1M NaCl，pH＝7.4)，继续在25℃室温下轻摇过夜。
(3)将步骤(2)的溶液离心(12000rpm，20min，15℃)去除多余的捕获序列，并用0.1M PBS溶液反复离心清洗三次。得到的捕获探针分散在储备溶液(10mM PB，0.1M NaCl，pH＝7.4)中。
制备的DNA功能化的纳米金捕获探针的紫外表征如图3所示。
图3：图中曲线a为金纳米粒子吸收光谱图，在520nm处出现了金纳米粒子的特征吸收峰，曲线b为捕获序列修饰上金纳米粒子后的吸收光谱图，其特征峰移动到了524nm处，即存在稍微红移，证实了捕获序列在金纳米粒子上的成功组装。
实施例2
取100μL实施例1制备的纳米金捕获探针溶液(探针终浓度为6nM)，分别向其中加入终浓度为200nM目标核酸，所述的目标核酸的序列是：SEQ ID NO.3：5’-AGTGATTTTAGAGAGAG-3’，室温下杂交10min后，加入终浓度为0.1U/μL的核酸外切酶III，37℃条件下反应30min。随后加入终浓度为15μg/mL的二硫化钼纳米片(尺寸200nm×200nm，二硫化钼的透射电镜图如图2)，静置反应70min，取上清液肉眼观察上清液颜色的变化，并通过紫外分光光度计测量上清液的吸光值变化。
实施例3
实施例3与实施例2方法相同，不同之处在于：金纳米粒子的粒径为10nm；目标核酸与捕获探针的杂交时间为30min；核酸外切酶III的终浓度为0.05U/μL，剪切时间为90min；二硫化钼纳米片的尺寸为100nm*100nm，终浓度为18μg/mL；静置反应时间为10min。
实施例4
实施例3与实施例2方法相同，不同之处在于：金纳米粒子的粒径为10nm；目标核酸与捕获探针的杂交时间为20min；核酸外切酶III的终浓度为0.2U/μL，剪切时间为20min；二硫化钼纳米片的尺寸为300nm*300nm，终浓度为5μg/mL；静置反应时间为90min。
实施例5
二硫化钼纳米片的孵育时间和孵育浓度对可视化检测的影响：
(1)取100μL实施例1制备的纳米金捕获探针溶液(探针终浓度为6nM)，分别向其中加入终浓度为200nM目标核酸，所述的目标核酸的序列是：SEQ ID NO.3：5’-AGTGATTTTAGAGAGAG-3’，室温下杂交10min后，加入终浓度为0.1U/μL的核酸外切酶III，37℃条件下反应30min。随后加入终浓度为15μg/mL的二硫化钼纳米片(尺寸200nm×200nm)，分别在0，10，20，30，40，50，60，70，80，90分钟时测量上清液的吸光度，探究不同的孵育时间对检测的影响，每组实验各重复3次。测试结果如图4所示：在0-70min内，随着时间推移，上清液的吸光度不断降低；当吸附时间达到70min时，MoS2对DNA-AuNPs探针的吸附达到平衡状态。因此，将MoS2与DNA-AuNPs的吸附时间优选为70min。
(2)取100μL实施例1制备的6nM纳米金捕获探针溶液(探针终浓度为6nM)，向其中加入终浓度为200nM目标核酸，室温下杂交10min后，加入终浓度为0.1U/μL的核酸外切酶III，37℃条件下反应30min。随后加入终浓度为分别为5，8，10，12，15，18μg/mL的二硫化钼纳米片(尺寸200nm×200nm)，静置70分钟后测量上清液的吸光度，探究不同的孵育浓度对检测结果的影响，每组实验各重复3次。测试结果如图5所示：当二硫化钼纳米片的孵育浓度为15μg/mL时，本检测方案得到了最高的信噪比。因此，将MoS2孵育浓度优选为15μg/mL。
实施例6
向本发明制备的可视化检测探针中加入不同浓度的目标核酸后，测试上清液的紫外可见吸收光谱随目标核酸浓度的变化实验：
取100μL实施例1制备的纳米金-捕获探针溶液(探针终浓度为6nM)，向其中分别加入终浓度分别为0nM，1nM，3nM，10nM，30nM，50nM，100nM，200nM，300nM的目标核酸，室温下杂交10min后，加入终浓度为0.1U/μL的核酸外切酶III，37℃条件下反应30min。随后加入终浓度为15μg/mL的二硫化钼纳米片(尺寸200nm×200nm)，静置70min后测量上清液的吸光度，每组实验各重复3次。测试结果如图6A所示：随着加入目标核酸浓度的增大，上清液吸光度逐渐增大，颜色从透明逐渐变为浅酒红色，说明本发明可以实现对不同浓度目标核酸的检测。图6B展示了上清液吸光度与一定浓度的目标核酸之间存在良好的线性关系(R2＝0.96364)，按照3倍标准差方法计算出检测限为0.59nM，说明检测方法具有良好的灵敏性。
实施例7
本发明制备的可视化探针对目标核酸检测的选择性实验：
各取100μL实施例1制备的纳米金-捕获探针溶液(探针终浓度为6nM)，向其中分别加入终浓度均为200nM的目标核酸DNA、与目标核酸单碱基错配的DNA1、双碱基错配的DNA2、三碱基错配的DNA3，室温下杂交10min后，加入终浓度为0.1U/μL的核酸外切酶III，37℃条件下反应30min。随后各加入终浓度为15μg/mL的二硫化钼纳米片，静置70min后测量上清液的吸光度，每组实验各重复3次。所述的与目标核酸碱基错配的序列分别是：DNA1:SEQ IDNO.4：5’-AGTGATTTAAGAGAGAG-3’；DNA2:SEQ ID NO.5：5’-AGA GAT TTA AGA GAG AG-3’；DNA3:SEQ ID NO.6：5’-AGA GAT TTA AGA GTG AG-3’。测试结果如图7所示：只有加入目标核酸后的上清液呈现酒红色，加入碱基错配的核酸反应后的上清液均接近透明。因此证明，本发明对目标核酸的检测具有良好的选择性。
实施例8
为验证本发明对于目标核酸检测具有通用性，针对随机目标核酸SEQ ID NO.7：5’-CACTTGAGGCTAACAC-3’，设计合理的纳米金-捕获探针进行检测。按实施例1所述方法和步骤制备纳米金-捕获探针，其捕获序列为：SEQ ID NO.8：5’-AAAAAAAAAAAAAAAAAAAATTTTTGTGTTAGCCTCAAGTG-3’(polyA修饰的捕获序列)或SEQ ID NO.9：5’-SH-TTTTTGTGTTAGCCTCAAGTG-3’(巯基修饰的捕获序列)。
各取100μL实施例6制备的6nM纳米金捕获探针溶液，分别向其中加入200nM目标核酸以及与目标核酸碱基错配的序列，所述的目标核酸的序列是：SEQ ID NO.7：5’-CACTTGAGGCTAACAC-3’，碱基错配的核酸序列是：SEQ ID NO.10：5’-CACTTGAAGCTAACAC-3’。按照实施例2所述进行反应。反应后取上清液肉眼观察上清液颜色的变化，并通过紫外分光光度计测量上清液的吸光值变化。测试结果如图8所示，只有加入目标核酸样本的上清液呈现浅酒红色，空白对照组以及加入碱基错配核酸样品的上清液均呈接近透明。该实施例证明，本发明对目标核酸的检测具有通用性，即通过合理的设计可对不同的目标核酸进行检测。
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Claims (9)

1.一种基于酶催化循环及二硫化钼介导吸附可视化检测核酸的方法，其特征在于，包括如下步骤：
(1)将巯基或polyA修饰的捕获序列组装在金纳米粒子表面，构建纳米金捕获探针；
(2)向含有目标核酸的待测体系中加入捕获探针，目标核酸与捕获探针完成杂交后向体系添加含有核酸外切酶III的反应液，进行循环剪切反应；
(3)向步骤(2)完成剪切反应后的体系中加入二硫化钼纳米片，静置反应；
(4)取步骤(3)反应后体系的上清液，肉眼观察上清液颜色的变化，并通过紫外分光光度计测量上清液的吸光值变化。
2.根据权利要求1所述的基于酶催化循环及二硫化钼介导吸附可视化检测核酸的方法，其特征在于，所述捕获序列通过加盐老化组装在金纳米粒子表面。
3.根据权利要求1所述的基于酶催化循环及二硫化钼介导吸附可视化检测核酸的方法，其特征在于，所述金纳米粒子的粒径优选为10-50nm。
4.根据权利要求2所述的基于酶催化循环及二硫化钼介导吸附可视化检测核酸的方法，其特征在于，所述加盐老化为将polyA或巯基修饰的目标核酸捕获序列与金纳米粒子混合，在室温下轻摇过夜；分批多次加入PBS溶液，继续室温下轻摇过夜；离心去除多余的捕获序列，离心清洗，得到的捕获探针分散在PBS溶液中储存。
5.根据权利要求1-4任一所述的基于酶催化循环及二硫化钼介导吸附可视化检测核酸的方法，其特征在于，所述捕获序列为与待检测目标核酸互补的序列。
6.根据权利要求1所述的基于酶催化循环及二硫化钼介导吸附可视化检测核酸的方法，其特征在于，步骤(2)所述目标核酸与捕获探针的杂交时间为10-30min。
7.根据权利要求1所述的基于酶催化循环及二硫化钼介导吸附可视化检测核酸的方法，其特征在于，步骤(2)所述循环剪切反应为采用核酸外切酶III进行，所述核酸外切酶III的终浓度为0.05-0.2U/μL，剪切时间为20-90min。
8.根据权利要求1所述的基于酶催化循环及二硫化钼介导吸附可视化检测核酸的方法，其特征在于，步骤(3)所述二硫化钼纳米片的尺寸为100-300nm*100-300nm，终浓度为5-18μg/mL。
9.根据权利要求1所述的基于酶催化循环及二硫化钼介导吸附可视化检测核酸的方法，其特征在于，步骤(3)所述静置反应时间为10-90min。
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Application publication date: 20191220

Assignee: NANJING YUANGAN MICROELECTRONIC CO.,LTD.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048124

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231127

Application publication date: 20191220

Assignee: Zhongkebaier (Nanjing) Biotechnology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048115

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231125

EE01 Entry into force of recordation of patent licensing contract
EE01 Entry into force of recordation of patent licensing contract

Application publication date: 20191220

Assignee: Nanjing Haihe Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048485

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231128

Application publication date: 20191220

Assignee: Xiaojiang Jiahu (Nanjing) Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048562

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231130

Application publication date: 20191220

Assignee: Xiaosu Accompanying Clinic (Nanjing) Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048561

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231130

Application publication date: 20191220

Assignee: Nanjing Youxin Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048560

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231130

Application publication date: 20191220

Assignee: Nanjing Zhimeng Rehabilitation Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048559

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231130

Application publication date: 20191220

Assignee: Qihe Technology (Nanjing) Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048558

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231129

Application publication date: 20191220

Assignee: Shuangqing Doctor Group (Hainan) Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048557

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231129

Application publication date: 20191220

Assignee: Shuangxin Internet Hospital (Hainan) Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048554

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231129

Application publication date: 20191220

Assignee: Xixiayuan (Ningxia) Agricultural Development Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048553

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231128

Application publication date: 20191220

Assignee: Nanjing Tongyou Engineering Services Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048552

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231128

Application publication date: 20191220

Assignee: Nanjing xinwindows Information Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048550

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231128

Application publication date: 20191220

Assignee: Nanjing Yangbang Enterprise Management Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048549

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231128

Application publication date: 20191220

Assignee: Nanjing Yixuntong Information Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048547

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231128

Application publication date: 20191220

Assignee: Nanjing Youda Medical Information Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048545

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231128

Application publication date: 20191220

Assignee: Nanjing Youda Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048541

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231128

Application publication date: 20191220

Assignee: Nanjing Hancai Electronics Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048538

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231128

Application publication date: 20191220

Assignee: Nanjing Hancai Optoelectronic Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048534

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231128

Application publication date: 20191220

Assignee: Nanjing Jianhai Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048527

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231128

Application publication date: 20191220

Assignee: Nanjing Qingyou Information Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048525

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231128

Application publication date: 20191220

Assignee: Nanjing Shuangzi Zhitong Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048523

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231128

Application publication date: 20191220

Assignee: NANJING PENGYUDA INFORMATION TECHNOLOGY CO.,LTD.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048517

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231128

Application publication date: 20191220

Assignee: Edge Intelligent Security Technology (Zhenjiang) Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048515

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231128

Application publication date: 20191220

Assignee: Edge Intelligence Research Institute Nanjing Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048511

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231128

Application publication date: 20191220

Assignee: Huiyi IoT Technology (Zhenjiang) Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048504

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231128

Application publication date: 20191220

Assignee: Jiangsu Hongyi Medical Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048500

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231128

Application publication date: 20191220

Assignee: Nanjing Aiqi Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980048488

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231128

EE01 Entry into force of recordation of patent licensing contract
EE01 Entry into force of recordation of patent licensing contract
EE01 Entry into force of recordation of patent licensing contract

Application publication date: 20191220

Assignee: Deloitte (Jiangsu) Education Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980049533

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231203

Application publication date: 20191220

Assignee: Nanjing Youqi Intelligent Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980049531

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231203

Application publication date: 20191220

Assignee: Nanjing Tuanyuan Intelligent Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980049522

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231203

Application publication date: 20191220

Assignee: Nanjing fandilang Information Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980049497

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231203

Application publication date: 20191220

Assignee: Dingshan Technology Incubation (Nanjing) Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980049483

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231203

Application publication date: 20191220

Assignee: Nanjing Jinxiang Experimental Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980049478

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231203

Application publication date: 20191220

Assignee: Nanjing Baoxing Intelligent Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980049437

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231203

Application publication date: 20191220

Assignee: Jiangsu Anbo Intelligent Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980049425

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231203

Application publication date: 20191220

Assignee: Nanjing Shihong Intelligent Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980049398

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231203

Application publication date: 20191220

Assignee: Nanjing Self Postal Transfer Technology Transfer Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980049391

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231203

Application publication date: 20191220

Assignee: Nanjing Lvran Agricultural Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980049370

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231203

Application publication date: 20191220

Assignee: Nanjing Huijue Intelligent Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980049366

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231203

Application publication date: 20191220

Assignee: Nanjing jinshuxin Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980049360

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231203

Application publication date: 20191220

Assignee: Nanjing Jingliheng Electronic Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980049351

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231203

Application publication date: 20191220

Assignee: Jiangsu Dixin Metrology Testing Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980049330

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231203

EE01 Entry into force of recordation of patent licensing contract

Application publication date: 20191220

Assignee: Nanjing yist Packaging Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980050260

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231207

EE01 Entry into force of recordation of patent licensing contract
EE01 Entry into force of recordation of patent licensing contract
EE01 Entry into force of recordation of patent licensing contract

Application publication date: 20191220

Assignee: Nanjing Shanyechu Agriculture and Forestry Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980051072

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231209

EE01 Entry into force of recordation of patent licensing contract

Application publication date: 20191220

Assignee: Jiangsu Liebao Network Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980052022

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231212

Application publication date: 20191220

Assignee: Jiangsu Chaoxin Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980052021

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231212

Application publication date: 20191220

Assignee: Speed Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980051704

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231212

Application publication date: 20191220

Assignee: Nanjing Zouma Information Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980051703

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231212

Application publication date: 20191220

Assignee: Nanjing Heyue Information Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980051698

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231212

Application publication date: 20191220

Assignee: Nantong Zhicheng Network Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980051315

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231212

EE01 Entry into force of recordation of patent licensing contract
EE01 Entry into force of recordation of patent licensing contract

Application publication date: 20191220

Assignee: Nanjing Shuhui Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980052024

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231213

Application publication date: 20191220

Assignee: Nanjing Qinghong Network Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980052023

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231213

Application publication date: 20191220

Assignee: Nanjing Aoweisen Gene Technology Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980051915

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231213

EE01 Entry into force of recordation of patent licensing contract
EE01 Entry into force of recordation of patent licensing contract

Application publication date: 20191220

Assignee: NANJING HUADONG ELECTRONICS VACUUM MATERIAL Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980053414

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231222

EE01 Entry into force of recordation of patent licensing contract
EE01 Entry into force of recordation of patent licensing contract
EE01 Entry into force of recordation of patent licensing contract

Application publication date: 20191220

Assignee: NANJING CREATCOMM TECHNOLOGY CO.,LTD.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980054276

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231227

Application publication date: 20191220

Assignee: NANJING YIZHIHENG SOFTWARE TECHNOLOGY Co.,Ltd.

Assignor: NANJING University OF POSTS AND TELECOMMUNICATIONS

Contract record no.: X2023980054071

Denomination of invention: A method for visualizing nucleic acid detection based on enzyme catalyzed cycling and molybdenum disulfide adsorption mediation

Granted publication date: 20221125

License type: Common License

Record date: 20231227