AAC Advances in Analytical Chemistry 2163-1557 Scientific Research Publishing 10.12677/aac.2024.142012 AAC-87303 aac2024142_71560398.pdf 化学与材料 基于核酸酶辅助信号放大的2’-O-甲基修饰分子信标用于高灵敏和特异性外泌体肿瘤microRNA分析 2’-O-Methyl-Modified Molecular Beacons for Highly Sensitive and Specific Exosomal Tumor microRNA Analysis Based on Nuclease-Assisted Signal Amplification 吕 天鹏 2 1 柯 声锋 2 1 王 书皓 2 1 崔 亮 2 1 浙江理工大学化学与化工学院，浙江 杭州 null 11 05 2024 14 02 95 105 © Copyright 2014 by authors and Scientific Research Publishing Inc. 2014 This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/

外泌体是新兴的重要癌症生物标志物。有效检测外泌体和外泌体内容物，特别是microRNA (miRNA)，对于癌症的诊断和治疗是迫切且具有挑战性的。基于双链特异性核酸酶(Duplex specific nuclease, DSN)的特异性识别和消化能力，我们设计了一个2’-O-甲基修饰的分子信标(2’-O-methyl-modified molecular beacon, omMB)，并开发了一个高灵敏度和特异性分析外泌体miRNA的信号放大检测平台。该方法以A375细胞分泌的外泌体为模型，以microRNA-21 (miR-21)为模型miRNA分子，可以检测到低至37.9 pM的miRNA和2 μg/mL的裂解外泌体。同时，与许多已开发的DSN辅助信号放大方法相比，新方法具有较高的特异性，可以区分错配miRNA。总之，这项工作为医学分析、临床应用和疾病诊断中的外泌体检测提供了一种有效的分析策略。 Exosomes are emerging important cancer biomarkers. Effective detection of exosome and exosomal contents, especially microRNA (miRNA), is urgent and challenging for cancer diagnosis and treatment. Based on specific recognition and digestion capacity of duplex specific nuclease (DSN), we designed a 2’-O-methyl-modified molecular beacon (omMB) and proposed an amplified detection platform for highly sensitive and specific analysis of exosomal miRNA. Taking A375 cell secreted exosomes as model targets and microRNA-21 (miR-21) as model miRNA molecules, the proposed method can detect miRNA down to 37.9 pM and 2 μg/mL lysed exosomes. Meanwhile, compared with many developed DSN-assisted amplified methods, the new method has high specificity which can distinguish mismatch miRNA. Overall, this work provides an effective analytical strategy for exosome detection in medical analysis, clinic applications and disease diagnosis.

 双链特异性核酸酶，外泌体，微小RNA，分子信标，信号放大, Duplex-Specific Nuclease Exosome MicroRNA Molecular Beacons Signal Amplification 摘要

外泌体是新兴的重要癌症生物标志物。有效检测外泌体和外泌体内容物，特别是microRNA (miRNA)，对于癌症的诊断和治疗是迫切且具有挑战性的。基于双链特异性核酸酶(Duplex specific nuclease, DSN)的特异性识别和消化能力，我们设计了一个2’-O-甲基修饰的分子信标(2’-O-methyl-modified molecular beacon, omMB)，并开发了一个高灵敏度和特异性分析外泌体miRNA的信号放大检测平台。该方法以A375细胞分泌的外泌体为模型，以microRNA-21 (miR-21)为模型miRNA分子，可以检测到低至37.9 pM的miRNA和2 μg/mL的裂解外泌体。同时，与许多已开发的DSN辅助信号放大方法相比，新方法具有较高的特异性，可以区分错配miRNA。总之，这项工作为医学分析、临床应用和疾病诊断中的外泌体检测提供了一种有效的分析策略。

 关键词

双链特异性核酸酶，外泌体，微小RNA，分子信标，信号放大

 2’-O-Methyl-Modified Molecular Beacons for Highly Sensitive and Specific Exosomal Tumor microRNA Analysis Based on Nuclease-Assisted Signal Amplification<sup> </sup>

Tianpeng Lyu, Shengfeng Ke, Shuhao Wang, Liang Cui*

School of Chemistry and Chemical Engineering, Zhejiang Sci-Tech University, Hangzhou Zhejiang

Received: Apr. 25th, 2024; accepted: May 15th, 2024; published: May 24th, 2024

 ABSTRACT

Exosomes are emerging important cancer biomarkers. Effective detection of exosome and exosomal contents, especially microRNA (miRNA), is urgent and challenging for cancer diagnosis and treatment. Based on specific recognition and digestion capacity of duplex specific nuclease (DSN), we designed a 2’-O-methyl-modified molecular beacon (omMB) and proposed an amplified detection platform for highly sensitive and specific analysis of exosomal miRNA. Taking A375 cell secreted exosomes as model targets and microRNA-21 (miR-21) as model miRNA molecules, the proposed method can detect miRNA down to 37.9 pM and 2 μg/mL lysed exosomes. Meanwhile, compared with many developed DSN-assisted amplified methods, the new method has high specificity which can distinguish mismatch miRNA. Overall, this work provides an effective analytical strategy for exosome detection in medical analysis, clinic applications and disease diagnosis.

Keywords:Duplex-Specific Nuclease, Exosome, MicroRNA, Molecular Beacons, Signal Amplification

Copyright © 2024 by author(s) and beplay安卓登录

This work is licensed under the Creative Commons Attribution International License (CC BY 4.0).

http://creativecommons.org/licenses/by/4.0/

 1. 引言

外泌体是一类由细胞分泌的细胞外囊泡，大小为30~150 nm [ 1 ] 。外泌体丰富且稳定，含有许多母体细胞的重要生物分子，如核酸(DNA、mRNA、miRNA)、蛋白质、糖及其它的大分子和小分子 [ 2 ] 。由于外泌体内容物(如蛋白质和miRNA)的水平与母体细胞直接相关，因此外泌体被认为是许多重要疾病如癌症的理想生物标志物 [ 3 ] [ 4 ] [ 5 ] 。在相关文献中已经报道了外泌体miRNA作为癌症直接相关的生物标志物。因此，对外泌体miRNA的分析成为疾病诊断、治疗监测和药物发现的迫切需要 [ 6 ] [ 7 ] [ 8 ] 。

目前，关于外泌体miRNA检测的工作已经有很多报道 [ 9 ] [ 10 ] [ 11 ] 。虽然逆转录PCR (qRT-PCR)、Northern Blot分析等传统检测方法表现出优异的分析性能，但在实际应用中仍存在成本高、操作复杂、灵敏度低等缺陷 [ 11 ] 。为了克服这些不足，许多等温的、核酸酶辅助的且灵敏度高的外泌体miRNA检测方法被相继报道 [ 12 ] ，包括聚合酶辅助的靶标扩增和核酸酶辅助的信号放大。以靶标miRNA为引物的聚合酶辅助的靶标扩增，如滚环扩增(rolling circle amplification, RCA)，可在聚合酶的帮助下形成长单链DNA [ 13 ] 。该方法可提高灵敏度，但耗时长，操作复杂。而核酸酶辅助的信号放大方法操作简单，设备便宜，更适合外泌体miRNA检测。例如，在之前的工作中，我们提出了一种氧化石墨烯(GO)保护的DNA探针用于miRNA分析，该探针依赖于DNase I和吸附在氧化石墨烯表面的单标记DNA荧光探针 [ 14 ] 。DNase I只能消化游离的DNA探针，不能消化RNA或氧化石墨烯保护的DNA探针。在目标miRNA存在的情况下，miRNA与氧化石墨烯保护的DNA探针杂交并从氧化石墨烯表面解吸，从而使DNA探针成为DNase I的底物并被消化。因此，自由的目标miRNA触发另外的混合–解吸–水解过程，使信号放大。该方法灵敏度高、选择性好、快速简便，可用于外泌体miRNA的检测 [ 15 ] [ 16 ] 。然而，由于这项工作需要纳米材料作为辅助，这增加了操作的复杂性，从而阻碍了其广泛应用。

相比之下，双特异性核酸酶(DSN)是等温扩增外泌体miRNA检测的较优方法。DSN可识别双链核酸，且只消化DNA。因此，它可以用于设计高灵敏的miRNA生物传感器。叶邦策教授课题组首次报道了以TaqMan探针作为报告信号的DSN辅助信号放大检测miRNA的方法。在这项工作中，miRNA与TaqMan探针杂交，DSN只能消化TaqMan探针从而释放miRNA。释放的miRNA可以与另一个TaqMan探针杂交，并导致其随后被消化。因此16，这种基于DSN的策略可以高灵敏度放大检测外泌体miRNA [ 17 ] 。基于其突出的特性，DSN已被广泛用于miRNA生物传感器的设计和外泌体miRNA分析。然而，作为线性探针，TaqMan探针的选择性较弱。例如，第一篇报道的基于DSN的信号放大分析方法无法区分单碱基错配的miRNA [ 18 ] [ 19 ] 。同时，荧光团和猝灭团分别标记在TaqMan探针的两端，距离较远会削弱FRET效率，增加背景信号，从而影响灵敏度。因此，传统的基于DSN的扩增方法需要提高灵敏度和选择性 [ 17 ] 。

在这项工作中，我们利用2’-O-甲基分子信标(omMB)的特异性序列识别能力和生物稳定性 [ 18 ] [ 20 ] [ 21 ] [ 22 ] ，提出了DSN辅助omMBs用于高灵敏度和特异性的外泌体miRNA分析。omMBs的发夹结构由于其固有的结构约束提高了特异性识别能力 [ 23 ] ，使该策略具有单碱基错配识别能力。因此，此信号放大方法可以检测低至37.9 pM的miRNA和2 μg/mL裂解外泌体。总之，该方法为医学分析、临床应用和疾病诊断中的外泌体检测提供了可能性。

 2. 材料和方法 2.1. 实验材料

DSN购自深圳新生物科技有限公司(中国深圳)。RNase抑制剂购自Takara有限公司(中国大连)。2’-O-甲基分子信标及miRNAs由生工生物科技有限公司(中国上海)合成，其序列列于表1。

Sequences used in this wor
Name Sequence (5’-3’)
2’-O-methyl MB FAM - CGA GC TCA ACA TCA GTC TGA TAA GCT A GC UCG - Dabcyl
MiR-21 UAG CUU AUC AGA CUG AUG UUG A
1-base mismatch UAG CUU AUC ACA CUG AUG UUG A
3-base mismatch UAG CUU AUC UUC CUG AUG UUG A

表1. 本工作中使用的序列

 2.2. 聚丙烯酰胺凝胶电泳分析

通过聚丙烯酰胺凝胶电泳(PAGE)检测DSN活性，5个Ep管中先加入10 μL DSN缓冲溶液(10 mM Tris-HCl (pH 8.0), 5 mM MgCl2and 1 mM DTT)，之后在5个Ep管中分别加入4 μM omMB (管1)，4 μM miR-21 (管2)，4 μM omMB + 0.25 U DSN (管3)，4 μM miR-21 + 0.25 U DSN (管4)，4 μM omMB + 4 μM miR-21 + 0.25 U DSN (管5)。5个样品在37℃下孵育1 h后加入10 μL饱和尿素停止反应，再加入4 μL 6x上样缓冲液后，样品可用于PAGE分析。

实验制备了15%变性的PAGE，其中含有4 ml 30% Acryl/Bis溶液(29:1)，1.4 ml H2O，3.3 g尿素和1.6 ml 5 × TBE。尿素溶解后，加入20 μL 10%过硫酸铵(APS)和20 μL四甲基乙二胺(TEMED)。充分混合后，在垂直平板电泳上加热30分钟后制成凝胶。之后，用Bio-Rad照射凝胶。

 2.3. 可行性分析

使用RF-5301-PC荧光分光光度计进行荧光测量(日本岛津)。激发光谱为485 nm，扫描波长为500~650 nm。为进行可行性分析，4个管中先加入100 μL DSN缓冲溶液，另外再分别加入100 nM omMB (管1)，100 nM omMB + 10 nM miR-21 (管2)，100 nM omMB + 0.25 U DSN (管3)，100 nM omMB + 10 nM miR-21 + 0.25 U DSN (管4)。37℃孵育1 h后使用荧光分光光度计进行分析。

 2.4. 灵敏度测试

制备DSN缓冲液2.1 mL，其中含omMB和DSN的浓度分别为100 nM和5.25 U。将缓冲液平均加入到21个Ep管中，21个样本分为3组(3个平行)。在1~7组样品中分别加入不同浓度的miR-21。37℃孵育1 h后，进行荧光测定。

 2.5. 细胞培养和外泌体分离

A375细胞系(人黑色素瘤)在Gibco Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA)中培养，添加10%胎牛血清和100 U/mL penicillin-streptomycin，在37℃和5% CO2环境下进行培养。为了排除外泌体的干扰，将它们培养在含有外泌体缺失的10%胎牛血清(FBS)的DMEM培养基中，胎牛血清(FBS)在100,000 g下超速离心18 h以去除FBS中的外泌体。然后将细胞培养上清液分别以300 g离心10分钟、2000 g离心20分钟、11,000 g离心45分钟，分别去除完整细胞、细胞碎片和蛋白质。随后，上清液以110,000 g 离心70 min获得沉积物外泌体。最后，沉淀物外泌体在PBS中重悬，保存于−80℃。

 2.6. 外泌体miRNA检测

为了检测外泌体miRNA，将外泌体95℃孵育10 min，然后16,500 g离心45 min，去除沉淀，收集上清。制备3组样品(每组8管反应缓冲液)后，在1到8组样品中分别加入不同浓度的外泌体。37℃孵育1 h后，通过荧光测量分析样品。

 3. 结果与讨论 3.1. 检测原理

图1. DSN辅助信号放大检测外泌体miRNA的工作原理

DSN可水解双链DNA或DNA/RNA杂交双链中的DNA，但对RNA无活性 [ 23 ] 。同时，2’-O-甲基修饰的双链可以抵抗核酸酶的酶切 [ 18 ] [ 24 ] 。本文合成了一个2’-O-甲基修饰的分子信标(omMB)：茎部由2’-O-甲基核苷酸组成，环部由正常脱氧核苷酸组成。如图1所示，在不存在目标miRNA的情况下，茎–环结构分子信标可以抵抗DSN消化并保持完整。在目标miRNA存在的情况下，形成分子信标/miRNA杂交双链，成为DSN的合适底物。DSN消化了MBs的环部分，并导致荧光恢复。与此同时，被释放的miRNA可与另一个omMB杂交，从而引发下一轮切割。因此，一个miRNA分子理论上可以通过循环过程触发无数omMB的水解，从而放大荧光信号。同时，由于omMBs对目标miRNA具有特异性识别能力，因此该方法可以区分单碱基错配序列。总的来说，所提出的策略可以有效地分析外泌体miRNA，具有高灵敏度和选择性。

 3.2. 可行性分析

在这项工作中，我们选择microRNA-21 (miR-21)作为模型靶标，这是一种重要的癌症生物标志物，据报道在许多肿瘤中高度过表达 [ 25 ] [ 26 ] 。随后，根据miR-21的序列设计相应的MB。首先通过聚丙烯酰胺凝胶电泳(PAGE)对该方法的可行性进行了评估。如图2(a)所示，通道1~4分别代表omMB、miR-21、omMB + DSN和miR-21 + DSN。在通道3中，与DSN孵育后，omMB仍然完好无损，这表明由于其茎由DNA双链组成，omMB可以抵抗DSN的消化，这可能是由于2’-O-甲基核苷酸的存在。通道4为miR-21 + DSN，由于DSN不能消化RNA，miR-21也如预期那样保持完整。然而，如通道5所示，当omMB与miRNA杂交时，miR-21保持完整，MB消失，说明MB在与miR-21杂交时被DSN消化，说明该方案的可行性。

在此基础上，我们通过荧光实验对比了有无DSN时的信号放大性能。如图2(b)所示，在没有DSN的情况下，100 nM MB在100 μL反应缓冲液中，溶液中的荧光强度较弱(黑线)。在反应缓冲液中加入10 nM的miR-21后，荧光信号增加1.4倍，表明miR-21与MB杂交成功。在DSN存在的情况下，MB的背景信号略有增加，这可能是由于omMB消化较弱造成的。然而，在加入miR-21后，由于DSN对MB的循环水解，观察到明显的2.1倍信号增强。这些结果表明，一个miR-21分子可以通过循环过程触发许多MB的水解，导致荧光信号放大。

图2. (a) omMB/miR-21双链上DSN活性的PAGE分析通道1：omMB；通道2：miR-21；通道3：omMB + DSN；通道4：miR-21 + DSN；通道5：omMB + miR-21 + DSN；(b) DSN辅助omMB检测miR-21的信号放大性能

 3.3. 温度优化

本工作的实验参数根据文献报道选择，如0.25 U DSN在100 μL 1x DSN反应缓冲液中孵育1 h。但是，文献报道的许多反应温度在50~60℃，在本实验中可能使omMB不稳定。这主要是由于omMB的发夹茎部会发生随机的构象变换，从而导致高背景信号，影响检测性能。为了达到最佳性能，对反应温度进行了评估。如图3所示，我们在含有100 nM omMB和0.25 U DSN的混合物中添加100 nM miR-21。之后分别在37℃、45℃和55℃下对其信噪比(SBR)进行比较。结果表明，该体系在37℃时的信噪比最好，这可能在omMB的结构稳定性和DSN的活性之间保持了良好的平衡。同时，我们也观察到部分文献也使用37℃作为反应温度 [ 27 ] [ 28 ] [ 29 ] 。

图3. 反应温度的优化A：37℃；B：45℃；C：55℃

 3.4. 检测灵敏度和选择性

图4. (a) 不同浓度miR-21的荧光光谱；(b) 不同浓度miR-21的荧光强度；(c) omMB辅助信号放大对miR-21检测的选择性分析F/F0：信号与背景比

在验证了该方法的可行性后，我们评估了在最佳实验条件下的检测灵敏度。图4(a)为加入不同浓度miRNA后记录的荧光信号。结果显示，随着miR-21浓度的增加，荧光信号逐渐增强。随着miR-21浓度的增加，从0到250 pM (见图4(b))，校准图呈线性，并通过公式拟合得很好：y = 54.21 + 104.28 x (n = 3, R2= 0.9937)，其中y为荧光信号，x为miR-21的浓度(nM)。计算得到的检出限为37.9 pM，与包括DSN辅助信号放大在内的多种核酸酶辅助信号放大方法相比具有一定的竞争力，具体比较如表2所示 [ 30 ] - [ 37 ] 。

Sensitivity comparison of miRNA analysis methods based on nucleas
Nuclease Detection limit Ref
Strand displacement (No nuclease) 462 pM (for miR-21) [ 30 ]
Molecular Beacon (No Nuclease) About 100 pM [ 31 ]
DSN 426 pM [ 32 ]
DSN 50 pM [ 33 ]
DNase I 9 pM [ 14 ]
DNase I 2.3 pM [ 34 ]
Exonuclease III 21.4 pM [ 35 ]
Exonuclease III 6 pM [ 36 ]
T7 Exonuclease 15 pM [ 37 ]
DSN 37.9 pM This work

表2. 基于核酸酶的miRNA分析方法的灵敏度比较

接下来，我们还测试了omMBs的选择性。如图4(c)所示，在MB体系中，miR-21的信号是1-mis miRNA的1.7倍，是3-mis miRNA的4.4倍，显示出高特异性，可以区分靶标miRNA与单碱基错配序列和三碱基错配序列。miRNA家族具有很高的同源性，区分密切相关的miRNA序列是一项具有挑战性的任务。由于其特异性水解，许多DSN辅助信号放大工作使用线性探针，如用于miRNA检测的TaqMan探针 [ 23 ] 。然而，TaqMan探针作为一种线性探针，对识别靶序列的选择性较弱 [ 38 ] ，如表3所示。我们的数据表明，通过使用omMB，DSN辅助信号放大系统可以对miRNA检测具有高选择性，这是相关性较强的miRNA序列分析的重要特征。

Selectivity of partial DSN auxiliary signal amplification technologie
Probes Single-base mismatch Specific Ref
Linear probe No data [ 23 ]
Linear probe No data [ 39 ]
Linear probe No data [ 40 ]
Linear probe No data [ 41 ]
Linear probe No data [ 42 ]
Linear probe No data [ 43 ]
Linear probe No data [ 44 ]
MB Yes [ 45 ]
G-triplex based MB Yes [ 46 ]
omMB Yes This work

表3. 部分的DSN辅助信号放大技术的选择性

 3.5. 外泌体miRNA检测与癌症诊断

在证明DSN辅助的2’-O-甲基扩增方法具有高灵敏度和选择性后，我们直接检测了A375外泌体中的miRNA，该外泌体已被证明具有miR-21的高表达 [ 7 ] 。分离并裂解A375外泌体后，检测加入不同浓度A375外泌体后的荧光强度。如图5所示，外泌体浓度的增加与荧光强度的增加是一致的。荧光强度在2.5~400 μg/mL范围内呈线性增长，检出限低至2 μg/mL。如表4所示，与已有检测线报道的扩增方法相比略低 [ 30 ] [ 31 ] [ 32 ] ，表明我们的策略对外泌体miRNA分析较为灵敏，这为之后区分正常及肿瘤患者提供了可能性。

图5. 荧光强度与外泌体浓度的工作曲线

Comparison of exosome detection line with other platform
Ref Detection limit
[ 30 ] 0.116 μg/mL
[ 31 ] 无具体的检测线
[ 32 ] 无具体的检测线
本文方法 2 μg/mL

表4. 与其它平台外泌体检测线的对比

 4. 结果与讨论

我们开发了一种DSN辅助的放大分析平台，使用2’-O-甲基分子信标作为报告探针，实现了高灵敏度和特异性的外泌体miRNA检测。2’-O-甲基修饰的分子信标具有发夹结构，能抵抗DSN的降解。该方法对miR-21的分析检测限(LOD)为37.9 pM，并且可以区分错配miRNA。然后，利用我们提出的方法检测裂解的外泌体，LOD为2 μg/mL。总之，所提出的策略简单、灵敏、选择性强。该平台可直接应用于临床，为疾病诊断和监测提供一种可行的方法。

 文章引用

吕天鹏,柯声锋,王书皓,崔 亮. 基于核酸酶辅助信号放大的2’-O-甲基修饰分子信标用于高灵敏和特异性外泌体肿瘤microRNA分析2’-O-Methyl-Modified Molecular Beacons for Highly Sensitive and Specific Exosomal Tumor microRNA Analysis Based on Nuclease-Assisted Signal Amplification[J]. 分析化学进展, 2024, 14(02): 95-105. https://doi.org/10.12677/aac.2024.142012

 参考文献 References Kalluri, R. and LeBleu, V.S. (2020) The Biology, Function, and Biomedical Applications of Exosomes. Science, 367, eaau6977.
https://doi.org/10.1126/science.aau6977 Selmaj, I., Cichalewska, M., Namiecinska, M., Galazka, G., Horzelski, W., Selmaj, K.W. and Mycko, M.P. (2017) Global Exosome Transcriptome Profiling Reveals Biomarkers for Multiple Sclerosis. Annals of Neurology, 81, 703-717.
https://doi.org/10.1002/ana.24931 Barile, L. and Vassalli, G. (2017) Exosomes: Therapy Delivery Tools and Biomarkers of Diseases. Pharmacology & Therapeutics, 174, 63-78.
https://doi.org/10.1016/j.pharmthera.2017.02.020 Cheng, N., Du, D., Wang, X., Liu, D., Xu, W., Luo, Y. and Lin, Y. (2019) Recent Advances in Biosensors for Detecting Cancer-Derived Exosomes. Trends in Biotechnology, 37, 1236-1254.
https://doi.org/10.1016/j.tibtech.2019.04.008 Stobiecka, M., Ratajczak, K. and Jakiela, S. (2019) Toward Early Cancer Detection: Focus on Biosensing Systems and Biosensors for an Anti-Apoptotic Protein Survivin and Survivin mRNA. Biosensors and Bioelectronics, 137, 58-71.
https://doi.org/10.1016/j.bios.2019.04.060 Drula, R., Ott, L.F., Berindan-Neagoe, I., Pantel, K. and Calin, G.A. (2020) MicroRNAs from Liquid Biopsy Derived Extracellular Vesicles: Recent Advances in Detection and Characterization Methods. Cancers, 12, Article 2009.
https://doi.org/10.3390/cancers12082009 Cui, L., Peng, R.X., Zeng, C.F., Zhang, J.L., Lu, Y.Z., Zhu, L., Huang, M.J., Tian, Q.H., Song, Y.L. and Yang, C.Y. (2022) A General Strategy for Detection of Tumor-Derived Extracellular Vesicle MicroRNAs Using Aptamer-Mediated Vesicle Fusion. Nano Today, 46, Article 101599.
https://doi.org/10.1016/j.nantod.2022.101599 Niu, Q., Gao, J., Zhao, K., Chen, X., Lin, X., Huang, C., An, Y., Xiao, X., Wu, Q., Cui, L., Zhang, P., Wu, L. and Yang, C. (2022) Fluid Nanoporous Microinterface Enables Multiscale-Enhanced Affinity Interaction for Tumor-Derived Extracellular Vesicle Detection. Proceedings of the National Academy of Sciences of the United States of America, 119, e2213236119.
https://doi.org/10.1073/pnas.2213236119 Jet, T., Gines, G., Rondelez, Y. and Taly, V. (2021) Advances in Multiplexed Techniques for the Detection and Quantification of MicroRNAs. Chemical Society Reviews, 50, 4141-4161.
https://doi.org/10.1039/D0CS00609B Wu, Y., Zhang, Y., Zhang, X., Luo, S., Yan, X., Qiu, Y., Zheng, L. and Li, L. (2021) Research Advances for Exosomal miRNAs Detection in Biosensing: From the Massive Study to the Individual Study. Biosensors and Bioelectronics, 177, Article 112962.
https://doi.org/10.1016/j.bios.2020.112962 Ouyang, T., Liu, Z., Han, Z. and Ge, Q. (2019) MicroRNA Detection Specificity: Recent Advances and Future Perspective. Analytical Chemistry, 91, 3179-3186.
https://doi.org/10.1021/acs.analchem.8b05909 Cheng, Y., Dong, L., Zhang, J., Zhao, Y. and Li, Z. (2018) Recent Advances in MicroRNA Detection. Analyst, 143, 1758-1774.
https://doi.org/10.1039/C7AN02001E Zhou, S., Sun, H., Dong, J., Lu, P., Deng, L., Liu, Y., Yang, M., Huo, D. and Hou, C. (2023) Highly Sensitive and Facile MicroRNA Detection Based on Target Triggered Exponential Rolling-Circle Amplification Coupling with CRISPR/ Cas12a. Analytica Chimica Acta, 1265, Article 341278.
https://doi.org/10.1016/j.aca.2023.341278 Cui, L., Lin, X., Lin, N., Song, Y., Zhu, Z., Chen, X. and Yang, C.J. (2012) Graphene Oxide-Protected DNA Probes for Multiplex MicroRNA Analysis in Complex Biological Samples Based on a Cyclic Enzymatic Amplification Method. Chemical Communications, 48, 194-196.
https://doi.org/10.1039/C1CC15412E Jin, D., Yang, F., Zhang, Y., Liu, L., Zhou, Y., Wang, F. and Zhang, G.J. (2018) ExoAPP: Exosome-Oriented, Aptamer Nanoprobe-Enabled Surface Proteins Profiling and Detection. Analytical Chemistry, 90, 14402-14411.
https://doi.org/10.1021/acs.analchem.8b03959 Wang, H., Chen, H., Huang, Z., Li, T., Deng, A. and Kong, J. (2018) DNase I Enzyme-Aided Fluorescence Signal Amplification Based on Graphene Oxide-DNA Aptamer Interactions for Colorectal Cancer Exosome Detection. Talanta, 184, 219-226.
https://doi.org/10.1016/j.talanta.2018.02.083 Wu, Y., Cui, S., Li, Q., Zhang, R., Song, Z., Gao, Y., Chen, W. and Xing, D. (2020) Recent Advances in Duplex-Specific Nuclease-Based Signal Amplification Strategies for MicroRNA Detection. Biosensors and Bioelectronics, 165, Article 112449.
https://doi.org/10.1016/j.bios.2020.112449 Lin, X.Y., Zhang, C., Huang, Y.S., Zhu, Z., Chen, X. and Yang, C.J. (2013) Backbone-Modified Molecular Beacons for Highly Sensitive and Selective Detection of MicroRNAs Based on Duplex Specific Nuclease Signal Amplification. Chemical Communications, 49, 7243-7245.
https://doi.org/10.1039/c3cc43224f Li, Y., Zhang, J., Zhao, J., Zhao, L., Cheng, Y. and Li, Z. (2016) A Simple Molecular Beacon with Duplex-Specific Nuclease Amplification for Detection of MicroRNA. Analyst, 141, 1071-1076.
https://doi.org/10.1039/C5AN02312B Gao, J.F., Li, Y., Li, W.Q., Zeng, C.F., Xi, F.N., Huang, J.H. and Cui, L. (2020) 2’-O-Methyl Molecular Beacon: A Promising Molecular Tool That Permits Elimination of Sticky-End Pairing and Improvement of Detection Sensitivity. RSC Advances, 10, 41618-41624.
https://doi.org/10.1039/D0RA07341E Zheng, H.Y., Lin, Q.Y., Zhu, J.C., Rao, Y.M., Cui, L., Bao, Y.Y. and Ji, T.H. (2021) DNase I-Assisted 2’-O-Methyl Molecular Beacon for Amplified Detection of Tumor Exosomal MicroRNA-21. Talanta, 235, Article 122727.
https://doi.org/10.1016/j.talanta.2021.122727 Sun, X., Ying, N., Ju, C., Li, Z., Xu, N., Qu, G., Liu, W. and Wan, J. (2018) Modified Beacon Probe Assisted Dual Signal Amplification for Visual Detection of MicroRNA. Analytical Biochemistry, 550, 68-71.
https://doi.org/10.1016/j.ab.2018.04.010 Yin, B.C., Liu, Y.Q. and Ye, B.C. (2012) One-Step, Multiplexed Fluorescence Detection of MicroRNAs Based on Duplex-Specific Nuclease Signal Amplification. Journal of the American Chemical Society, 134, 5064-5067.
https://doi.org/10.1021/ja300721s Tsourkas, A., Behlke, M.A. and Bao, G. (2002) Hybridization of 2’-O-Methyl and 2’-Deoxy Molecular Beacons to RNA and DNA Targets. Nucleic Acids Research, 30, 5168-5174.
https://doi.org/10.1093/nar/gkf635 Wang, Y., Gao, X., Wei, F., Zhang, X., Yu, J., Zhao, H., Sun, Q., Yan, F., Yan, C., Li, H. and Ren, X. (2014) Diagnostic and Prognostic Value of Circulating miR-21 for Cancer: A Systematic Review and Meta-Analysis. Gene, 533, 389-397.
https://doi.org/10.1016/j.gene.2013.09.038 Calin, G.A. and Croce, C.M. (2006) MicroRNA Signatures in Human Cancers. Nature Reviews. Cancer, 6, 857-866.
https://doi.org/10.1038/nrc1997 Wang, Q., Yin, B.C. and Ye, B.C. (2016) A Novel Polydopamine-Based Chemiluminescence Resonance Energy Transfer Method for MicroRNA Detection Coupling Duplex-Specific Nuclease-Aided Target Recycling Strategy. Biosensors and Bioelectronics, 80, 366-372.
https://doi.org/10.1016/j.bios.2016.02.005 Yang, C., Dou, B., Shi, K., Chai, Y., Xiang, Y. and Yuan, R. (2014) Multiplexed and Amplified Electronic Sensor for the Detection of MicroRNAs from Cancer Cells. Analytical Chemistry, 86, 11913-11918.
https://doi.org/10.1021/ac503860d Yuan, Y.H., Chi, B.Z., Wen, S.H., Liang, R.P., Li, Z.M. and Qiu, J.D. (2018) Ratiometric Electrochemical Assay for Sensitive Detecting MicroRNA Based on Dual-Amplification Mechanism of Duplex-Specific Nuclease and Hybridization Chain Reaction. Biosensors and Bioelectronics, 102, 211-216.
https://doi.org/10.1016/j.bios.2017.11.030 Wang, H., He, D., Wan, K., Sheng, X., Cheng, H., Huang, J., Zhou, X., He, X. and Wang, K. (2020) In situ Multiplex Detection of Serum Exosomal MicroRNAs Using an All-in-One Biosensor for Breast Cancer Diagnosis. Analyst, 145, 3289-3296.
https://doi.org/10.1039/D0AN00393J Lee, J.H., Kim, J.A., Kwon, M.H., Kang, J.Y. and Rhee, W.J. (2015) In situ Single Step Detection of Exosome MicroRNA Using Molecular Beacon. Biomaterials, 54, 116-125.
https://doi.org/10.1016/j.biomaterials.2015.03.014 Gao, Z., Yuan, H., Mao, Y., Ding, L., Effah, C.Y., He, S., He, L., Liu, L.E., Yu, S., Wang, Y., Wang, J., Tian, Y., Yu, F., Guo, H., Miao, L., Qu, L. and Wu, Y. (2021) In situ Detection of Plasma Exosomal MicroRNA for Lung Cancer Diagnosis Using Duplex-Specific Nuclease and MoS2 Nanosheets. Analyst, 146, 1924-1931.
https://doi.org/10.1039/D0AN02193H Liu, H., Fan, J.L., Liu, W.P., Tong, C.Y., Xie, Z.H., Deng, R.L. and Long, X.Y. (2018) A Dual Signal Amplification Method for miR-204 Assay by Combining Chimeric Molecular Beacon with Double-Stranded Nuclease. Analytical Methods, 10, 5834-5841.
https://doi.org/10.1039/C8AY02147C Xie, Y., Lin, X.Y., Huang, Y.S., Pan, R.J., Zhu, Z., Zhou, L.J. and Yang, C.Y.J. (2015) Highly Sensitive and Selective Detection of miRNA: DNase I-Assisted Target Recycling Using DNA Probes Protected by Polydopamine Nanospheres. Chemical Communications, 51, 2156-2158.
https://doi.org/10.1039/C4CC08912J Tang, Y.F., Liu, M.X., Xu, L.C., Tian, J.N., Yang, X.L., Zhao, Y.C. and Zhao, S.L. (2018) A Simple and Rapid Dual-Cycle Amplification Strategy for MicroRNA Based on Graphene Oxide and Exonuclease III-Assisted Fluorescence Recovery. Analytical Methods, 10, 3777-3782.
https://doi.org/10.1039/C8AY01106K Liu, M.X., Liang, S.P., Tang, Y.F., Tian, J.N., Zhao, Y.C. and Zhao, S.L. (2018) Rapid and Label-Free Fluorescence Bioassay for MicroRNA Based on Exonuclease III-Assisted Cycle Amplification. RSC Advances, 8, 15967-15972.
https://doi.org/10.1039/C8RA01605D Wei, K.J., Zhao, J.J., Qin, Y.F., Li, S.T., Huang, Y. and Zhao, S.L. (2018) A Novel Multiplex Signal Amplification Strategy Based on Microchip Electrophoresis Platform for the Improved Separation and Detection of MicroRNAs. Talanta, 189, 437-441.
https://doi.org/10.1016/j.talanta.2018.07.037 Bonnet, G., Tyagi, S., Libchaber, A. and Kramer, F.R. (1999) Thermodynamic Basis of the Enhanced Specificity of Structured DNA Probes. Proceedings of the National Academy of Sciences of the United States of America, 96, 6171-6176.
https://doi.org/10.1073/pnas.96.11.6171 Lu, Y.Y., Wang, L. and Chen, H.Q. (2019) Turn-on Detection of MicroRNA155 Based on Simple UCNPs-DNA-AuNPs Luminescence Energy Transfer Probe and Duplex-Specific Nuclease Signal Amplification. Spectrochimica Acta Part A: Molecular Spectroscopy, 223, Article 117345.
https://doi.org/10.1016/j.saa.2019.117345 Li, Y.T., Tang, D.H., Zhu, L., Cai, J.T., Chu, C.N., Wang, J., Xia, M., Cao, Z.Z. and Zhu, H. (2019) Label-Free Detection of miRNA Cancer Markers Based on Terminal Deoxynucleotidyl Transferase-Induced Copper Nanoclusters. Analytical Biochemistry, 585, Article 113346.
https://doi.org/10.1016/j.ab.2019.113346 Liu, Q., Kang, P.J., Chen, Z.P., Shi, C.X., Chen, Y. and Yu, R.Q. (2019) Highly Specific and Sensitive Detection of MicroRNAs by Tandem Signal Amplification Based on Duplex-Specific Nuclease and Strand Displacement. Chemical Communications, 55, 14210-14213.
https://doi.org/10.1039/C9CC06790F Degliangeli, F., Kshirsagar, P., Brunetti, V., Pompa, P.P. and Fiammengo, R. (2014) Absolute and Direct MicroRNA Quantification Using DNA-Gold Nanoparticle Probes. Journal of the American Chemical Society, 136, 2264-2267.
https://doi.org/10.1021/ja412152x Tan, L., Xu, L., Liu, J.W., Tang, L.J., Tang, H. and Yu, R.Q. (2019) Duplex-Specific Nuclease-Mediated Target Recycling Amplification for Fluorescence Detection of MicroRNA. Analytical Methods, 11, 200-204.
https://doi.org/10.1039/C8AY02265H Peng, W.P., Zhao, Q., Piao, J.F., Zhao, M., Huang, Y.W., Zhang, B., Gao, W.C., Zhou, D.M., Shu, G.M., Gong, X.Q. and Chang, J. (2018) Ultra-Sensitive Detection of MicroRNA-21 Based on Duplex-Specific Nuclease-Assisted Target Recycling and Horseradish Peroxidase Cascading Signal Amplification. Sensors and Actuators B: Chemical, 263, 289-297.
https://doi.org/10.1016/j.snb.2018.02.143 Xiao, M.S., Chandrasekaran, A.R., Ji, W., Li, F., Man, T.T., Zhu, C.F., Shen, X.Z., Pei, H., Li, Q. and Li, L. (2018) Affinity-Modulated Molecular Beacons on MoS2 Nanosheets for MicroRNA Detection. ACS Applied Materials & Interfaces, 10, 35794-35800.
https://doi.org/10.1021/acsami.8b14035 Wu, Z.F., Zhou, H., He, J., Li, M., Ma, X.M., Xue, J., Li, X. and Fan, X.T. (2019) G-Triplex Based Molecular Beacon with Duplex-Specific Nuclease Amplification for the Specific Detection of MicroRNA. Analyst, 144, 5201-5206.
https://doi.org/10.1039/C9AN01075K
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