Journal of Sensor Technology and Application
Vol.05 No.04(2017), Article ID:22289,10 pages
10.12677/JSTA.2017.54007

A Novel Fluorescent Biosensor for Ultrasensitive Detection of miRNA-122 Based on Rolling Circle Amplification Coupled with Nicking Enzyme Signal Amplification

Xu Wang1, Chenggong Xu2, Jingfeng Wang2, Yu Wang2

1Shandong Yellow River Institute of Metrology, Jinan Shandong

2School of Biological Sciences and Technology, University of Jinan, Jinan Shandong

Received: Sep. 20th, 2017; accepted: Oct. 5th, 2017; published: Oct. 12th, 2017

ABSTRACT

Aberrant expression of microRNA-122 (miRNA-122) is associated with the occurrence, development and metastasis of hepatocellular carcinoma. It’s of great importance for the development of sensitive and selective method for the detection of miRNA-122 to the early diagnosis and prognosis of hepatocellular carcinoma. A novel fluorescent biosensor has been constructed for ultrasensitive and high specific detection of miRNA-122 based on rolling circle amplification (RCA) coupled with nicking endonuclease signal amplification (NESA). Our assay involves of two stages of reaction mechanism, the first stage is target-activated nicking endonuclease-assisted polymerization, which is used for the realization of the recycle amplification of target and secondary target and the produce of RCA primer. The second stage is to achieve signal transduction and multiple signal amplification by the utilization of RCA coupled with NESA. The results reveal the constructed biosensor display high sensitivity and specificity for miRNA-122 detection, and the limit of detection is as low as 3.9 aM, which is significantly improved compared to the previous methods. Moreover, the proposed method has the advantages of rapidness, low cost, simplicity with only one-step operation. Therefore, the developed RCA coupled with NESA-based fluorescent biosensor might create a simple and practical platform for the detection of miRNA and the early diagnosis and prognosis of cancer.

Keywords:Rolling Circle Amplification, Nicking Endonuclease Signal Amplification, Fluorescence Biosensor, MiRNA-122

基于滚环扩增技术耦合切刻内切酶信号放大技术的新型超灵敏miRNA-122荧光传感器

王煦1,徐成功2,王敬峰2,王玉2

1山东省黄河计量研究院,山东 济南

2济南大学生物科学与技术学院,山东 济南

收稿日期:2017年9月20日;录用日期:2017年10月5日;发布日期:2017年10月12日

摘 要

MicroRNA-122 (miRNA-122)的异常表达与肝癌的发生、发展及转移紧密相关,发展简单、快速、高灵敏和高特异性的miRNA-122检测方法对于肝癌的早期诊断和预后评估具有非常重要的意义。本文报道了一种基于滚环扩增技术(RCA)耦合切刻内切酶信号放大(NESA)技术的新型荧光传感器并用于超灵敏和高选择性检测miRNA-122。该方法涉及了两个阶段的反应机制,第一阶段,目标物激活切刻内切酶辅助的聚合反应,用以实现目标物与次级目标物循环放大以及生成RCA引物;第二阶段,利用RCA耦合NESA反应,实现信号传导以及多重信号放大。实验结果证明,该传感器表现出对miRNA-122的非常高的灵敏度和特异性,检测下限达到3.9 aM,较之前报道的方法,灵敏度有明显的提高。另外,该方法整个过程只需一步反应,具有操作简便、成本低、检测时间短的优势。所以,所提出的基于RCA耦合NESA的荧光传感器有望建立一个简单而实用的生物传感新平台,并应用于miRNA检测以及癌症的早期诊断和预后评估。

关键词 :滚环扩增技术,切刻内切酶信号放大,荧光传感器,miRNA-122

Copyright © 2017 by authors and Hans Publishers Inc.

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

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

1. 引言

miRNA-122是科学家们发现的一种非编码特异性的多聚腺苷酸RNA,其前体定位于人类第18号染色体18q21.31位点上 [1] 。在肝脏的生长发育过程中,miRNA-122对其生理、病理作用的应用广泛,在体内呈持续表达 [2] 。有研究表明,miRNA-122参与了肝细胞发育以及肝细胞应急应答等一系列基本生命过程 [3] 。病理状态下,miRNA-122能够促进丙型肝炎病毒(HCV)在肝细胞内复制,可以说,miRNA-122与肝癌(HCC)的发生、发展及转移紧密相关 [4] [5] 。所以,发展简单、快速、高灵敏和高特异性的miRNA-122检测方法对于肝癌的早期诊断和预后评估具有非常重要的意义。

近几十年里,科学家们一直致力于发展新型高效的miRNA检测技术。已经发展起来的检测方法主要包括Northern杂交 [6] 、实时荧光定量PCR [7] 和DNA微阵列 [8] 等,虽然这些方法在核酸鉴定和定量检测领域发挥了重要的作用,然而,他们也各自有一些不足之处。例如,对于Northern杂交,整个操作过程较为复杂、耗时长,且灵敏度较低,无法满足人们对miRNA的高灵敏检测的需求;对于实时荧光定量PCR方法,检测过程中样品容易受到污染,并且,整个操作过程需要昂贵的设备和经过专门培训的技术人员,因此,其实用性并不是很好;对于DNA微阵列技术,分析成本较高、灵敏度有限。因此,亟需发展操作简单、检测时间短、成本低且具有超高灵敏度和特异性的新型miRNA检测方法。

为满足痕量分析物的高灵敏检测需求,利用核酸扩增技术对目标物的量进行放大是一种有效的方法。近来,科学家们提出了一些高效的核酸等温扩增技术,如环介导等温扩增技术(LAMP) [9] 、滚环扩增技术(RCA) [10] [11] 、链置换扩增技术(SDA) [12] 、切刻内切酶信号放大(NESA) [13] [14] 以及外切酶信号放大技术 [15] [16] 等等,这些技术已经被广泛地应用于核酸、蛋白质、小分子以及细胞等的检测研究 [17] [18] [19] 。本文中,我们结合RCA技术具有的高扩增效率以及NESA技术的高特异性的特点,发展了一种简单、快速、低成本的新型荧光生物传感方法并用于miRNA-122的超灵敏和高选择性检测。该方法包括两个阶段的反应过程,第一阶段,目标物激活内切酶辅助的聚合反应,用以实现目标物与次级目标物循环放大以及生成RCA引物;第二阶段,利用RCA耦合NESA反应,实现信号传导以及多重信号放大。基于所提出的多重放大策略,该传感器能够实现miRNA-122的超灵敏和高特异性检测。实验结果显示,该传感器在最优实验条件下,对miRNA-122的检测下限达到3.9 aM。另外,该方法整个过程只需一步反应,操作简便、成本低、检测时间短,无需昂贵的仪器和专门的技术人员。所以,所提出的基于RCA耦合NESA的荧光传感器有望建立一个简单而实用的生物传感新平台,并应用于miRNA检测以及癌症的早期诊断和预后评估。

2. 实验部分

2.1. 材料与试剂

实验中所用到的phi29 DNA聚合酶、限制性核酸内切酶(Nb.BbvCI)、T4 DNA连接酶、核酸外切酶Ⅰ、核酸外切酶Ⅲ、dNTPs以及相应的反应缓冲液均购买于New England Biolabs公司(中国,北京)。RNA酶抑制剂和焦磷酸二乙酯(DEPC)购买于宝生物工程有限公司(中国,大连)。所有其它的化学试剂均是分析纯,购买于国药集团化学试剂有限公司(北京,中国)。实验所用的溶液均由超纯水配制,其电阻值 > 18.26 MΩ/cm。实验所用的缓冲溶液和超纯水均需灭菌处理。所有的核酸探针均由生工生物工程有限公司(中国上海)合成,其序列由表1所示。

Table 1. The nucleic acid sequence used in the experiment

表1. 实验中所用到的核酸序列

注:挂锁探针的5'端修饰有磷酸基团;分子信标的5'端修饰有FAM荧光基团,3'端修饰有DABCYL猝灭基团。

2.2. 环状模板制备

环状模板的制备过程如下所述,首先,将1 μL 连接探针(100 μM)、1 μL挂锁探针(100 μL)和1 μL T4 DNA连接酶(60 U μL−1)加入到T4 DNA连接酶缓冲液中,充分混匀后,在16℃条件下培育12小时。然后,将反应溶液在65℃条件下培育15分钟,使T4 DNA连接酶变性失活,再将1 μL核酸外切酶Ⅰ(20 U μL−1)和1 μL核酸外切酶Ⅲ(20 U μL−1)加入到反应溶液中,目的是水解消化体系中游离的连接探针和挂锁探针以及结合在环形模板DNA上的连接探针,以获得纯净的环形模板。接下来,再将反应溶液在85℃条件下培育10分钟以使核酸外切酶Ⅰ和核酸外切酶Ⅲ失活。最后,将环形模板保存在4℃冰箱备用。

2.3. miRNA-122检测

将1 μL不同浓度的目标物miRNA-122加入到19 μL含有1 μL发夹型结构探针(0.1μM)、1 μL辅助探针(0.1 μM)、1 μL环形模板(0.1 μM)、1 μL分子信标(100 μM)、1 μL phi29 DNA聚合酶、4 μL dNTPs (250 μM)和1 μL Nb.BbvCI的反应溶液中,充分混匀后,在37℃条件下培育1小时。反应结束后,将上述反应溶液在85℃条件下培育10分钟,使体系中所用到的酶变性失活。利用Cary Eclipse荧光仪(Agilent)对反应后的样品进行荧光检测,激发波长为486 nm,发射波长范围为500 nm~600 nm,激发狭缝和发射狭缝为5 nm。

3. 结果与讨论

3.1. 传感器的工作原理

我们所构建的荧光传感器的工作原理如图1所示,首先,设计了两条独特的核酸探针,分别是包含有发夹型结构探针(hairpin probe, HAP)3'末端互补序列、Nb.BbvCI识别序列和RCA引物互补序列的辅助探针(help probe),以及包含有目标物miRNA-122互补序列、限制性内切酶Nb.BbvCI识别序列和辅助探针互补序列的发夹型结构探针。当体系中存在目标物miRNA-122时,miRNA-122与HAP的5'末端发生特异性的识别反应,导致发夹型结构探针被打开,从而辅助探针的5'末端可以与暴露出来的3'末端退火杂交,在phi29 DNA聚合酶和dNTP的作用下,辅助探针的3'凹端作为引物,以HAP为模板进行延伸反应,致使miRNA-122从HAP上脱离下来,并且,生成了包含有Nb.BbvCI的识别位点的双链DNA,Nb.BbvCI能够对双链DNA的特定位点进行切割,从而发生“延伸–切割”循环放大反应,生成了与miRNA-122具有相同序列的次级目标物(miDNA-122),并且,解离下来的miRNA-122以及所生成的miDNA-122可以与其它的HAP发生杂交反应,引发新的“延伸–切割”循环放大反应,使miRNA-122获得指数级的扩增;同时,当辅助探针通过碱基互补配对作用结合到HAP上来时,HAP的3'凹端作为引物,以辅助探针为模板进行延伸反应,由于辅助探针序列中包含有Nb.BbvCI识别序列和RCA引物互补序列,故Nb.BbvCI能够对所生成的双链DNA的特定位点进行切割,从而发生“延伸–切割”循环放大反应,产生了大量的RCA引物。随后,在环形模板(circular template)、phi29 DNA聚合酶和dNTPs的参与下,发生RCA反应,生成了非常多的包含有大量重复序列的单链DNA。由于环形模板序列中包含了与分子信标(molecule beacon, MB)相同的序列,故生成的RCA产物能够与大量的MB退火杂交。另外,由于分子信标序列中包含Nb.BbvCI识别序列,故Nb.BbvCI能够对结合到RCA产物上来的MB进行切割,被切成两段的MB无法稳定地与RCA产物结合,从RCA产物上脱离下来,从而RCA产物可以与其它的MB退火杂交,引发新一轮的“退火–切割”反应,导致大量的MB被切断,从而释放出极强的荧光信号。基于上述的RCA耦合NESA的多重循环放大机理,该传感器有望实现对

Figure 1. Schematic illustration of fluorescent biosensor for miRNA assay based on RCA coupled with NESA

图1. 基于RCA耦合NESA的miRNA荧光传感器的原理示意图

目标物miDNA-122的超灵敏和高特异性检测。

3.2. 可行性验证

为了验证实验方案的可行性,我们进行了一系列的控制实验,实验结果如图2所示。图2是该传感器在不同条件下用于miRNA-122检测所获得的荧光光谱图。我们观察到,空白样品具有非常弱的荧光信号,这归因于分子信标中荧光基团和猝灭基团足够接近,能够发生荧光共振能量转移,导致荧光基团的荧光被有效地猝灭(黑色曲线)。相反地,对于阳性样品,呈现出非常强的荧光信号,说明目标物miRNA-122激活了内切酶辅助的目标循环放大反应以及RCA反应,生成了大量的RCA产物,使无数的MB被切断而释放出非常强的荧光信号(红色曲线)。当反应体系中不存在Nb.BbvCI或者phi29 DNA聚合酶时,我们观察到不太明显的荧光峰,这表明RCA引物的产生依赖于Nb.BbvCI (蓝色曲线)和phi29 DNA聚合酶(黄色曲线)协同催化的循环切割反应。另外,当使用非目标物miRNA-205取代miRNA-122时,荧光光谱呈现非常弱的荧光信号,说明荧光信号的释放是由特异性的目标识别反应引发的,而不是其他非特异性的因素(绿色曲线)。以上的实验数据证实所构建的传感器能够用于miRNA-122检测。

3.3. 实验条件优化

考虑到MB浓度和反应时间对方法的信背比有一定的影响,我们对其进行了优化。通过在不同条件下,比较传感器用于阳性样品和空白样品检测所获得的荧光强度比值,来获得最佳的实验条件。图3是使用不同浓度的MB所对应的荧光强度比值,从图中可以看出,随着反应体系中MB浓度的逐渐增加,方法的信背比(F/F0)也逐渐增加,当MB浓度达到5 μM时,方法的信背比反而减小,所以,5 μM被确定为最佳的MB浓度。图4是不同反应时间条件所对应的荧光强度值,可以看出,阳性样品的荧光强度值不断升高,当反应时间达到60 min时,荧光强度值变化不大,而空白样品随反应时间增多,荧光强度值变化不太明显,所以,我们选择60 min作为最合适的反应时间。

Figure 2. Fluorescence spectra responses of the biosensor for miRNA-122 assay. Blank sample (black curve), positive sample (red curve, the concentration of miRNA-122 is 1 pM), miRNA-122 (1 pM) + HAP + helper probe + phi 29 + dNTPs + MB (blue curve), miRNA-122 (1 pM) + HAP + helper probe + dNTPs + Nb.BbvCI + MB (yellow curve), miRNA-205 (1 nM) + HAP + helper probe + dNTPs + Nb.BbvCI + MB (green curve)

图2. miRNA-122检测的荧光光谱图。空白样品(黑色线),阳性样品(miRNA-122浓度为1 pM,红色线),miRNA-122 (1 pM) + HAP + helper probe + phi 29 + dNTPs + MB (蓝色线),miRNA-122 (1 pM) + HAP + helper probe + dNTPs + Nb.BbvCI + MB (黄色线),miRNA-205 (1 nM) + HAP + helper probe + dNTPs + Nb.BbvCI + MB (绿色线)

Figure 3. Effect of the concentration of MB on the fluorescence signal intensity. F and F0 represent the fluorescence signal intensity at 518 nmin the presence (the black pillars) and absence (the red pillars) of miRNA-122, respectively. The concentrations of MB are 0.5 μM, 1 μM, 5 μM and 10 μM, respectively. Error bars are standard deviations across three repetitive experiments

图3. 不同浓度分子信标条件下miRNA检测的荧光响应。F和F0分别表示传感器用于阳性样品(黑色带)和空白样品(红色带)检测时的荧光强度值。分子信标的浓度分别是,MB1:0.5 μM;MB2:1 μM;MB3:5 μM;MB4:10 μM。纵坐标是波长518 nm处的荧光强度值。误差棒为三次测量的标准偏差

Figure 4. Effect of the reaction time on the fluorescence signal intensity. The red curve and black curve represent the fluorescence signal intensity at 518 nm in the presence and absence of miRNA-122, respectively. Error bars are standard deviations across three repetitive experiments

图4. 不同反应时间条件下miRNA检测的荧光响应。红色线和黑色线分别表示传感器用于阳性样品和空白样品检测时的荧光强度值。纵坐标是波长518 nm处的荧光强度值。误差棒为三次测量的标准偏差

3.4. 特异性考察

为考察所构建荧光传感器的选择性,我们将其用于目标miRNA-122以及四种非目标miRNA (miRNA-205, miRNA-155, miRNA-21和let-7a)的定量检测,所获得的荧光信号如图5所示。由图中可以看出,几种非目标miRNA获取的的荧光信号与空白样品的荧光信号非常接近,说明该传感器对于miRNA-122检测具有非常高的特异性。

3.5. miRNA-122检测的工作曲线

在最佳的实验条件下,我们对不同浓度目标物miRNA-122 (0 aM,10 aM,100 aM,1 fM,10 fM,100 fM,1 pM)的荧光响应进行了考察,结果如图6所示。由图6(a)中可以看出,随着miRNA-122浓度的逐渐增加,所获得的荧光光谱的荧光峰强度逐渐增强。图6(b)给出了波长518 nm处的荧光强度值与miRNA-122浓度对数(lg)值的线性关系,该校准曲线拟合方程为F = 140.97143X − 47.73333(F表示波长518 nm处获得的荧光强度值,X表示miRNA-122浓度的对数值),其相关系数为0.996。由图6(b)可以看出,传感器用于miRNA-122检测的线性范围是10 aM到1 pM,检测下限达3.9 aM,与之前报道的方法 [20] [21] [22] [23] 比较,我们的方法具有更宽的响应范围和更高的灵敏度。

4. 结论

本文构建了一种基于滚环扩增技术耦合切刻内切酶信号放大技术的新型miRNA-122荧光传感器,该方法涉及聚合酶和切刻内切酶辅助的目标物循环放大和次级目标物循环放大、滚环复制放大以及切刻内切酶辅助的分子信标循环放大等多重放大策略。实验结果表明,该传感器表现出对miRNA-122的非常高的特异性,其用于miRNA-122检测的线性范围是10 aM到1 pM,检测下限达3.9 aM,与之前报道的方法比较,灵敏度具有显著的提高。并且,该方法整个过程只需一步反应,具有操作简便、成本低、检测

Figure 5. Fluorescence responses at 518 nm of our biosensor to different miRNA. The concentration of miR-122 is 1 pM. The concentration of non-target miRNA is 1 nm. Error bars are standard deviations across three repetitive experiments

图5. 不同种类miRNA检测的荧光响应。其中,miRNA-122的浓度为1 pM,其他四种miRNA (miRNA-205,miRNA-155,miRNA-21和let-7a)的浓度为1 nM。纵坐标是波长518 nm处的荧光强度值。误差棒为三次测量的标准偏差

(a)(b)

Figure 6. (a) Fluorescence emission spectra responses to different concentrations of miR-122. (b) The calibration curve of fluorescence intensity at 518 nm for different miR-122 concentrations. Error bars are standard deviations across three repetitive experiments

图6. (a) 不同浓度miRNA-122检测的荧光光谱图。(b) 波长在518 nm处的荧光强度值与miRNA-122浓度的线性关系图。误差棒为三次测量的标准偏差

时间短的优势。因此,我们发展的基于滚环扩增技术耦合切刻内切酶信号放大技术的荧光生物传感技术有望为miRNA-122检测以及相关的癌症临床早期诊断和预后评估提供一种简单而实用的新工具。

文章引用

王煦,徐成功,王敬峰,王玉. 基于滚环扩增技术耦合切刻内切酶信号放大技术的新型超灵敏miRNA-122荧光传感器
A Novel Fluorescent Biosensor for Ultrasensitive Detection of miRNA-122 Based on Rolling Circle Amplification Coupled with Nicking Enzyme Signal Amplification[J]. 传感器技术与应用, 2017, 05(04): 53-62. http://dx.doi.org/10.12677/JSTA.2017.54007

参考文献 (References)

  1. 1. Jopling, C.L., Norman, K.L. and Sarnow, P. (2006) Positive and Negative Modulation of Viral and Cellular mRNAs by Liver-Specific microRNA miRNA-122. Cold Spring Harbor Symposia on Quantitative Biology, 71, 369-376. https://doi.org/10.1101/sqb.2006.71.022

  2. 2. Shu, X.L., Fan, C.B., Long, B., Zhou, X. and Wang, Y. (2016) The Anti-Cancer Effects of Cisplatin on Hepatic Cancer Are Associated with Modulation of miRNA-21 and miRNA-122 Expression. European Review for Medical and Pharmacological Sciences, 20, 4459-4465.

  3. 3. Bandopadhyay, M., Sarkar, N., Datta, S. and Das, D. (2016) A Pal Hepatitis B virus X Protein Mediated Suppression of miRNA-122 Expression Enhances Hepatoblastoma Cell Proliferation through Cyclin G1-p53 Axis. Infectious Agents and Cancer, 11, 40. https://doi.org/10.1186/s13027-016-0085-6

  4. 4. Girard, M., Jacquemin, E., Munnich, A., et al. (2008) miRNA-122, a Paradigm for the Role of microRNA in the Liver. Journal of Hepatology, 48, 648-659. https://doi.org/10.1016/j.jhep.2008.01.019

  5. 5. Tsai, W.C., Hsu, P.W., Lai, T.C., et al. (2009) MicroRNA-122, A Tumor Suppressor microRNA that Regulates Intrahepatic Metastasis of Hepatocellular Carcinoma. Hepatology, 49, 1571-1582. https://doi.org/10.1002/hep.22806

  6. 6. Dallman, M.J., Porter, A.C., Larsen, C.P., et al. (1989) Lymphokine Production in Al-lografts-Analysis of RNA by Northern Blotting. Transplantation Proceedings, 21, 296-298.

  7. 7. Kwok, S. and Higuchi, R. (1989) Avoiding False Positives with PCR. Nature, 339, 237-238.

  8. 8. Zhou, H., Chen, Q., Tan, W. Qiu, Z. and Li, S. (2017) Integrated Clinicopathological Features and Gene Microarray Analysis of Pancreatic Neuroendocrine Tumors. Gene, 625, 72-77. https://doi.org/10.1016/j.gene.2017.05.009

  9. 9. Mori, Y. and Notomi, T. (2009) Loop-Mediated Isothermal Amplification (LAMP): A Rapid, Accurate, and Cost-Effective Diagnostic Method for Infectious Diseases. Journal of Infection and Chemotherapy, 15, 62-69. https://doi.org/10.1007/s10156-009-0669-9

  10. 10. Zhang, Y., Yang, L., Lin, C., Guo, L. et al. (2015) Fluorescence Aptasensor for Ochratoxin A in the Food Samples Based on Hyperbranched Rolling Circle Amplification. Analytical Methods, 53, 250-252. https://doi.org/10.1039/C5AY01182E

  11. 11. Brasino, M.D. and Cha, J.N. (2015) Isothermal Rolling Circle Amplification of Virus Genomes for Rapid Antigen Detection and Typing. Analyst, 140, 5138-5144. https://doi.org/10.1039/C5AN00721F

  12. 12. Lee, S.J., Cho, Y.H., Kim, C.S., et al. (2004) Screening for Chlamydia and Gonorrhea by Strand Displacement Amplification in Homeless Adolescents Attending Youth Shelters in Korea. Journal of Korean Medical Science, 19, 495-500. https://doi.org/10.3346/jkms.2004.19.4.495

  13. 13. Wen, Y.Q., Xu, Y., Mao, X.H., Wei, Y.L., Song, H.Y., Chen, N., Huang, Q., Fan, C.H. and Li, D. (2012) DNAzyme-Based Rolling-Circle Amplification DNA Machine for Ultrasensitive Analysis of MicroRNA in Drosophila Larva. Analytical Chemistry, 84, 7664-7669. https://doi.org/10.1021/ac300616z

  14. 14. Zhao, Y.X., Chen, F., Li, Q., Wang, L.H. and Fan, C.H. (2015) Isothermal Amplification of Nucleic Acids. Chemical Reviews, 115, 12491-12545. https://doi.org/10.1021/acs.chemrev.5b00428

  15. 15. Wang, M., Fu, Z.L., Li, B.C., Zhou, Y.L., Yin, H.S. and Ai, S.Y. (2014) One-Step, Ultrasensitive, and Electrochemical Assay of MicroRNAs Based on T7 Exonuclease Assisted Cyclic Enzymatic Amplifi-cation. Analytical Chemistry, 86, 5606-5610. https://doi.org/10.1021/ac5010376

  16. 16. Shi, X.M., Fan, G.C., Shen, Q.M. and Zhu, J.J. (2016) Photoelectrochemical DNA Biosensor Based on Dual-Signal Amplification Strategy Integrating Inorganic-Organic Na-nocomposites Sensitization with λ-Exonuclease-Assisted Target Recycling. ACS Applied Material & Interfaces, 8, 35091-35098. https://doi.org/10.1021/acsami.6b14466

  17. 17. Deng, R.J., Zhang, K.X. and Li, J.H. (2017) Isothermal Amplification for Mi-croRNA Detection: From the Test Tube to the Cell. Accounts of Chemical Research, 50, 1059-1068. https://doi.org/10.1021/acs.accounts.7b00040

  18. 18. Zhuang, J.Y., Lai, W.Q., Xu, M.D., Zhou, Q. and Tang, D.P. (2015) Plas-monic AuNP/g-C3N4 Nanohybrid-Based Photoelectrochemical Sensing Platform for Ultrasensitive Monitoring of Polynucleotide Kinase Activity Accompanying Dnazyme-Catalyzed Precipitation Amplification. ACS Applied Material & Interfaces, 7, 8330-8338. https://doi.org/10.1021/acsami.5b01923

  19. 19. Qian, Y., Fan, T.T., Wang, P., Zhang, X., Luo, J.J., Zhou, F.Y., et al. (2017) A Novel Label-Free Homogeneous Electrochemical Immunosensor Based on Proximity Hybridization-Triggered Isothermal Exponential Amplification Induced G-Quadruplex Formation. Sensors and Actuators B: Chemical, 248, 187-194. https://doi.org/10.1016/j.snb.2017.03.152

  20. 20. Yang, C.-T., Pourhassan-Moghaddam, M., Wu, L., Bai, P. and Thierry, B. (2017) Ultrasensitive Detection of Cancer Prognostic miRNA Biomarkers Based on Surface Plasmon Enhanced Light Scattering. ACS Sensors, 2, 635-640. https://doi.org/10.1021/acssensors.6b00776

  21. 21. Lv, S.F., Chen, F., Chen, C.Y., Chen, X.M., Gong, H. and Cai, C.Q. (2017) A Novel CdTe Quantum Dots Probe Amplified Resonance Light Scattering Signals to Detect MicroRNA-122. Talanta, 165, 659-663. https://doi.org/10.1016/j.talanta.2017.01.020

  22. 22. Bi, S., Chen, M., Jia, X.Q. and Dong, Y. (2015) A Hot-Spot-Active Magnetic Graphene Oxide Substrate for MicroRNA Detection Based on Cascaded Chemiluminescence Resonance Energy Transfer. Nanoscale, 7, 3745-3753. https://doi.org/10.1039/C4NR06603K

  23. 23. Bi, S., Yue, S.Z., Song, W.L. and Zhang, S.S. (2016) A Target-Initiated DNA Net-work Caged on Magnetic Particles for Amplified Chemiluminescence Resonance Energy Transfer Imaging of MicroRNA and Targeted Drug Delivery. Chemical Communication, 52, 12841-12844. https://doi.org/10.1039/C6CC05187A

期刊菜单