CN108445067B - 一种双信号无酶的信号放大rna纳米生物传感器、制备方法及其应用 - Google Patents
一种双信号无酶的信号放大rna纳米生物传感器、制备方法及其应用 Download PDFInfo
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Abstract
本发明提供了一种双信号无酶的信号放大RNA纳米生物传感器、制备方法及其应用,与现有技术相比,本发明为了放大电化学响应,使用“ΔIMB+ΔIFc”作为响应信号来定量测定miRNA浓度。与现有技术相比,与现有技术相比,本发明提供的电化学传感器的灵敏度高,选择性高,检测限低,不需要复杂的操作步骤和样品预处理,操作简单,且可以很快的得到结果;仪器小、轻便、廉价,花费小;电极尺寸小,传质速率快,检测更灵敏。
Description
技术领域
本发明属于生物传感器领域,具体涉及一种双信号无酶的信号放大RNA纳米生物传感器、制备方法及其应用。
背景技术
RNA(Ribonucleic Acid)存在于生物细胞中以及部分病毒、类病毒中,是由核糖核苷酸分子经磷酸二酯键缩聚而成的长链分子。一个核糖核苷酸分子由磷酸,核糖和碱基构成。RNA 是基因表达的关键调控因子,参与调控细胞周期、细胞增殖、分化、凋亡、代谢和转录。
MicroRNA(miRNA)是一类包含18-25个核苷酸长的非编码内源性RNA单链。虽然这些分子是非编码的,但它们在基因表达中起着至关重要的作用。它们可以通过与mRNA的3'非翻译区的互补结合来调节植物和动物中基因表达。
最近,证明了miRNA在癌症中起重要作用。不规则的miRNA表达和许多疾病有关联,例如神经障碍、肿瘤、心血管和自身免疫性疾病。miRNA在癌细胞中的表达失调、在癌症细胞中变异的miRNA表达的独特模式可以作为诊断指纹。因此,在多原发性和转移性癌症中miRNA 的表达水平被用于构建能够通过其原发部位识别癌症转移的miRNA分类器。两种最有特点的肿瘤抑制miRNA是miR-15和miR-16。B细胞慢性淋巴细胞白血病(CLL)是发达国家最常见的成人白血病,与染色体区域的丢失有关。该区域包含miR-15和miR-16,其在约70%的CLL 患者中丢失。因此,miRNA是癌症生物标志物的良好候选物,并且可能预示诊断,预后和预测。
RNA生物标志物的研究发展迅速,涌现了大量高性能的RNA检测方法。这些方法大多基于分子生物学技术,如定量逆转录聚合酶链反应(RT-qPCR)、微阵列和RNA测序。但这些方法通常须要一些繁琐的样品预处理步骤和昂贵的仪器。此外,为了避免样本的异质性,在体液中检测RNA生物标记物需要相对较大的样本容量。为获得相对稳健、准确和有效的方法,产生了几种基于纳米技术的RNA传感方法,包括光学和电化学方法。这些方法取样程序是比较简单的,能进行快速的分析。其中,电化学方法已经达到了超高灵敏度和选择性,具有多路复用分析的潜力。电化学RNA传感器的问题仍然存在,例如室温下RNA通常不稳定、易降解, RNA的制备和提取困难,生物环境复杂,RNA的长度各异、短RNA的检测困难等。
纳米技术的快速发展引领了新型生物传感技术的发展。例如光学传感器、纳米孔传感器、电化学生物传感器等。通常,一个有效的生物传感器主要由两个部分组成:一个受体(它专门识别目标分析物)和识别生物分子相互作用的传感器(信号生成和增强元素),并将这种相互作用转化为信号进行测量。电化学RNA传感器大多是基于靶RNA序列与电极表面结合的互补受体(主要是DNA寡核苷酸)的杂交。用探针对RNA进行杂交,可以得到一个可测量的电化学信号。然后,对RNA的检测主要通过伏安法、安培法和阻抗法来进行。与此同时,也使用基于芯片的纳米结构微电极对RNA生物标志物进行多路复用检测,并开发出了少量的超灵敏电化学方法。发夹DNA探针是生物标志物筛选中用途最广泛的寡核苷酸探针之一,通常用于RNA 检测的光学和电化学分析。
目前,RNA检测技术步骤繁琐、耗时,仪器昂贵,测定复杂、灵敏度低等。在体液中检测RNA生物标记物需要相对较大的样本容量。室温下RNA通常不稳定、易降解,生物环境复杂,RNA的长度各异。且miRNA的长度短,检测困难。
发明内容
本发明的目的在于提供一种双信号无酶的信号放大RNA纳米生物传感器及其制备方法,灵敏度高,选择性高,检测限低,成本低。
本发明还提供了一种双信号无酶的信号放大RNA纳米生物传感器检测miRNA-16的应用。
本发明具体技术方案如下:
本发明提供的一种双信号无酶的信号放大RNA纳米生物传感器的制备方法,包括以下步骤:
1)将探针1P-MB与DTT缓冲溶液混合均匀后,静置;然后加入乙酸溶液和乙酸钠溶液,再加入乙醇,混合,离心,除去上清液,干燥,即得处理后的探针1;
2)将步骤1)处理后的探针1用缓冲溶液溶解后,将金纳米线电极浸入探针1缓冲溶液中,孵育后,用巯基己醇封闭,得MB/NWE电极;
3)再将MB/NWE电极置于探针2P-Fc缓冲溶液中,杂交,探针2与探针1通过碱基对的互补配对,得Fc/MB/NWE电极,即为双信号无酶的信号放大RNA纳米生物传感器。
步骤1)具体为:
将1OD的探针1与100uL DTT缓冲溶液混合均匀后,静置;然后加入200uL质量浓度6%的乙酸溶液和50uL 3M的乙酸钠溶液,再加入100%乙醇1.6mL,混匀,6000r/min转下离心5分钟,除去上清液,35℃恒温环境下通风一夜,干燥,即得处理后的探针1;
步骤1)探针1P-MB序列:5’-HS-SH-C6-TGG CAG CAC ATT GCC G-C6-MB-3’;
所述DTT缓冲溶液的制备方法为:
4mg DTT用100uL 0.1M pH8.5的tris-HCl缓冲溶液溶解。
步骤1)中所述静置时间为2-3h,优选在室温下静置。
步骤2)具体为:
将步骤1)处理后的探针1用0.01M的pH7.4的PBS缓冲溶液溶解,稀释到5uM;将金纳米线电极浸入探针1缓冲溶液中,恒温37℃下孵育8-12小时;然后室温下将电极浸入100uL浓度10uM的巯基己醇溶液中,封闭2小时,得MB/NWE电极。因探针1一端连有巯基,金硫键作用将探针1接在电极上。修饰完后使用巯基己醇封闭两小时,使P-MB链有序,用蒸馏水洗去未修饰的探针。探针1的一端连有亚甲基蓝,且探针1易于形成颈环结构,在0.01M的PBS溶液中扫SWV(方波伏安法)。
步骤2)中所述金纳米线电极的制备方法为:
将直径25um的2cm金微米线装入长度8cm、内径0.64mm、外径1mm毛细管中央,在P-2000 激光拉制仪下加热并水平拉断,在真空状态下,加热温度400-460℃,加热四个循环,每个循环加热15-25s,冷却35-45s,使得毛细管与金属丝熔融包裹严实;然后将加热温度设置为 400-460℃,拉力130-150N,速率45-55m/s,在该条件下拉断,之后使用钨丝连通导电,经过磨擦金面露出,即为金纳米盘电极,用氢氟酸刻蚀金纳米盘电极,获得金纳米线电极。
步骤3)具体为:
将MB/NWE电极置于含2uM探针2P-Fc的0.01M PBS溶液中,恒温37℃下杂交8-12小时,探针2与探针1通过碱基对的互补配对,使探针1的颈环结构解开形成较稳定的双链结构阻碍亚甲基蓝的电子传递,得到Fc/MB/NWE电极,即为双信号无酶的信号放大RNA纳米生物传感器。
所述探针2P-Fc序列:5’-CGC CAA TAT TTA CGT GCT GCT A-C6-Fc-3’;
还包括步骤4):将该生物传感器与miRNA-16杂交,验证该传感器对目标RNA的特异性灵敏响应。
步骤4)具体为:将购来的miRNA-16目标物,加入无菌水配成2uM的miRNA-16溶液,37℃下将Fc/MB/NWE电极与200uL 2uM的miRNA-16溶液杂交8-12小时,以验证所构建生物传感器对RNA-16的灵敏响应。杂交后的电极在含有5mM MgCl2和140mM NaCl的0.01M PBS 缓冲溶液中浸泡处理,然后再检测,由于高离子强度下,Mg离子有利于核糖核苷酸的互补配对(杂交和解链)。
所述miRNA-16目标物序列:5’-UAG CAG CAC GUA AAU AUU GGC G-3’;
进一步的,步骤4)中所述PBS缓冲溶液pH范围在6-8,优选为7.5。
本发明提供的一种双信号无酶的信号放大RNA纳米生物传感器,采用上述方法制备得到。
本发明提供的一种双信号无酶的信号放大RNA纳米生物传感器检测miRNA-16的应用,具体检测方法为:
将制备的双信号无酶的信号放大RNA纳米生物传感器与不同浓度的miRNA-16杂交,杂交后的电极在含有5mM MgCl2和140mM NaCl的0.01M PBS中处理后,检测SWV响应,构建杂交前后MB和Fc峰电流变化值之和与miRNA-16的浓度的对数的线性关系,实现对miRNA-16的检测。
进一步的,miRNA-16的浓度分别为100fM、1pM、10pM、100pM、1nM、10nM和100nM。
所述得到的线性关系为:(ΔIMB+ΔIFc)(pA)=296.38+21.059lgCmiRNA(M)(R2=0.9916)。
与现有技术相比,本发明为了放大电化学响应,使用“ΔIMB+ΔIFc”作为响应信号来定量测定miRNA浓度。与现有技术相比,本发明提供的电化学传感器的灵敏度高,选择性高,检测限低,不需要复杂的操作步骤和样品预处理,操作简单,且可以很快的得到结果;仪器小、轻便、廉价,花费小;电极尺寸小,传质速率快,检测更灵敏。
附图说明
图1为本发明检测原理图;
图2为金纳米盘电极和刻蚀后的金纳米线电极在5mM二茂铁乙腈溶液中的CV,扫速为 50mV/s;
图3为金纳米线电极在5mM二茂铁乙腈溶液中的循环伏安曲线(A线)与COMSOL模拟数据(B线)的对比图,电极半径15nm,长40nm,扫速为50mV/s;
图4A为MB/NWE电极在0.01M PBS溶液中的方波伏安响应;
图4B为Fc/MB/NWE电极在0.01M PBS溶液中的方波伏安响应;
图4C为与目标miRNA杂交后的Fc/MB/NWE电极在0.01M PBS溶液中的方波伏安响应;
图5为不同pH所对应的峰电流;
图6A为不同浓度的miRNA-16处理Fc/MB/NWE电极后的SWV响应;a-h目标物浓度为0、 100fM、1pM、10pM、100pM、1nM、10nM、100nM;
图6B为MB和Fc峰值电流与miRNA浓度的对数的线性拟合曲线;miRNA浓度从100fM到 100nM;
图7为MB和Fc峰电流变化值之和与miRNA浓度的对数的线性关系,miRNA浓度从100fM 到100nM;
图8为对不同碱基序列的RNA的特异性响应。由上至下分别为空白,完全错配RNA,多碱基错配RNA,单碱基错配RNA,miRNA-16;不同序列的RNA浓度均为2uM;单碱基错配RNA序列:5’-UAG CAG CAC GCA AAU AUU GGC G-3’;多碱基错配RNA序列:5’-UUG UAG UAC ACAAAA AUA GUG G-3’;完全错配RNA序列:5’-AGU UCA GGU CUU GGC GAA CAU C-3’;
图9为与目标杂交前和被目标杂交解链后组装探针二的RNA生物传感器的电化学响应;
图10为Fc/MB/NWE电极在连续七天测量的SWV响应。
具体实施方式
实施例1
一种双信号无酶的信号放大RNA纳米生物传感器的制备方法,包括以下步骤:
1)取4mg DTT用100uL 0.1M,pH8.5的tris-HCl缓冲溶液充分溶解,制成DTT缓冲溶液;将1OD探针1P-MB溶液与DTT缓冲溶液混合均匀后,室温静置3h;然后加入200uL质量浓度6%的乙酸溶液和50uL 3M的乙酸钠溶液,在加入100%乙醇1.6mL,混匀,6000r/min 离心,除去上清液,35℃恒温环境下通风一夜,干燥,即得处理后的探针1;
2)将步骤1)处理后的探针1用0.01M的pH7.4的PBS溶解,稀释到5uM,将以激光拉制法制备的直径60nm的金纳米线电极浸入其中,恒温37℃孵育12h后,然后室温下将处理后的电极浸入100uL浓度10uM的巯基己醇溶液中,封闭2小时,得MB/NWE电极,因探针1一端连有巯基,金硫键作用将探针一接在电极上。修饰完后使用巯基己醇封闭两小时,使P-MB 链有序,用蒸馏水洗去未修饰的探针。探针1的一端连有亚甲基蓝,且探针一易于形成颈环结构,在0.01M的PBS溶液中扫SWV。具体条件为:在配有三电极系统的CHI660D电化学工作站上进行,纳米电极作为工作电极,铂(Pt)丝作为辅助(对)电极,银/氯化银(Ag/AgCl) 电极作为参比电极。其电位设置为0.5V至-0.05V,频率25Hz,振幅0.025mV,结果如图4A,在-0.28V附近有较强的亚甲基蓝信号,表明带亚甲基蓝的DNA单链成功接在电极上,此为 MB/NWE电极。
3)再将MB/NWE电极浸入含2uM探针2P-Fc的0.01M PBS溶液,恒温37℃下杂交12小时,探针2与探针1通过碱基对的互补配对,得Fc/MB/NWE电极;探针2与探针1通过碱基对的互补配对,使探针1的颈环结构解开形成较稳定的双链结构阻碍亚甲基蓝的电子传递。如图4B在PBS中的SWV在0.18V处附近出现较强的Fc峰,且-0.28V处MB的峰信号减小,说明Fc/MB/NWE电极制备成功,即为双信号无酶的信号放大RNA纳米生物传感器。
4)将该生物传感器与1uM miRNA-16杂交,杂交后的电极在含有5mM MgCl2和140mMNaCl 的0.01M PBS缓冲溶液中浸泡处理,以方波伏安法进行检测。其SWV如图4C,-0.28V处峰信号增强,0.18V处信号减小。说明制备的的双标记信号放大RNA传感器对目标物具有灵敏响应。
步骤2)中所述金纳米线电极的制备方法为:
将直径25um的2cm金微米线装入长度约8cm、内径0.64mm、外径1m的毛细管中央,在P-2000激光拉制仪下加热并水平拉断。在真空状态下,加热温度400-460℃,加热四个循环,每个循环加热15-25s,冷却35-45s,使得毛细管与金属丝熔融包裹严实。然后将加热温度设置为400-460℃,拉力130-150N,速率45-55m/s,在该条件下拉断。拉断之后仪器会显示拉制时间,一般在3-4s为正常。将这两段具有超细尖端的毛细管在显微镜下观察;可看到直径小于100纳米的金丝被包裹在尖锐的尖端中,之后使用钨丝连通导电。经过磨擦金面露出,即为金纳米盘电极,用氢氟酸刻蚀金纳米盘电极,获得金纳米线电极。使用刻蚀的方法制备了尺寸不同的金纳米线电极。金纳米线的长度可由如下公式来进行计算:
iqss是极限电流,n是每摩尔分子转移电子数目,F是法拉第常数,D是扩散系数,Cb是溶液的浓度,r0是电极半径,A为金纳米线电极的表面积。
图2是半径约15nm的纳米盘电和经1:4(V/V)的氢氟酸中刻蚀后的电极的二茂铁表征图。刻蚀前是半径约15nm的金纳米盘电极,在5mM二茂铁(乙腈)中的循环伏安曲线呈现很好的 S型。在氢氟酸溶液中刻蚀10s,将外层玻璃刻蚀掉,得到了图2中刻蚀后的曲线。与盘电极比较,刻蚀后的电极其稳态伏安电流明显增大。通过comsol模拟软件可以得出刻蚀后的纳米线(红色)长约40nm。图3是该金纳米线电极的实验数据(A线)与模拟数据(B线)的对比图。模拟数据是在半径15nm、长40nm的纳米线,在扫速50mV/s、溶液为5mM二茂铁的条件下,得到的稳态伏安响应。可以看出,实验与模拟的结果是完全吻合的,说明使用的模拟软件是正确的、合理的,是能够与实验数据拟合的。模拟数据的循环伏安曲线没有充电电流,在电位反扫时能够与氧化过程完全重合,呈现完美的“S”型。而实验结果的充电电流也很小,说明制备的纳米线电极很成功。
上述制备的MB/NWE电极、Fc/MB/NEW电极和制得的双信号无酶的信号放大RNA纳米生物传感器与目标miRNA-16杂交后在0.01M PBS溶液中的方波伏安响应。结果如图4A、图4B和图4C。
溶液是pH值对实验的结果有显著影响,主要是影响生物传感器中RNA的活性和指示小分子MB和Fc的活性。改变步骤4)中所用PBS检测液的pH值,观察方波伏安响应的峰强度变化。溶液pH范围在6~8之间变化,分别作SWV。由于-0.28V处的峰变化很小,故以0.18V处的峰值电流为对象,从图3可以看出,当pH为7.5时,还原峰最大,因此pH7.5时为最优条件,如图5。
本发明检测原理如图1所示,在没有目标物存在时,金纳米电极的表面发生有两个氧化还原反应发生,但双链结构阻碍了亚甲基蓝分子的电子传递,二茂铁分子更容易传递电子,就出现了图中b曲线。当目标物与其中一条链结合时,将该链从电极上解离下来,带亚甲基蓝分子的RNA链恢复单链结构,在Mg离子的存在下,形成颈环结构,MB的电化学信号增强,如图a曲线。实验使用的是方波伏安法(SWV),电位区间为0.5V至-0.5V,目标RNA的检测是通过分析亚甲基蓝还原峰电流的增大值和二茂铁还原峰电流的减小值之和。
生物传感器选择性的研究:
为了验证上述制备的传感器对miRNA-16具有特异性响应,选择了四种不同碱基序列的 RNA,浓度均为2uM,杂交时间12h。如图8,分别为空白,完全不互补的RNA,多碱基错配的 RNA,单碱基错配的RNA和完全互补的miRNA-16。可以看出在没有RNA存在的空白中,亚甲基蓝峰较小,二茂铁峰较大;与完全不互补的RNA杂交后,在PBS中的SWV与空白相比几乎没有变化;与多碱基错配的RNA配对后,其电化学信号变化值约为目标物的10%;而单碱基错配的RNA其电化学信号的变化约为30%。这个结果显示本发明制备的传感器的选择性优越。
生物传感器再生性的研究:
传感器的可再生性是评价传感器性能的重要指标,决定着传感器可否重复利用。已知 Fc/MB/NWE电极与目标杂交可使探针二与探针一解链。如图9所示,A是与目标杂交之前的 Fc/MB/NWE电极,其双链结构影响亚甲基蓝分子的电子传递,形成A的曲线。在于目标杂交后,双链被解开,将于目标杂交后的电极,浸入2uM的探针二溶液中孵育12小时,得到了B 的曲线。在解链重组前后,电极的重现性很好。说明本发明制备的电极是可再生的、可重复利用的。
生物传感器稳定性的研究:
图10是本发明制备的RNA生物传感器在连续七天内在pH7.5的0.01M PBS溶液中的电化学响应,实验检测后将传感器置于冰箱中冷藏。可以看出亚甲基蓝和二茂铁的信号大小有轻微的有降低。这种情况出现的原因可能是RNA和标记小分子的活性在低温下随时间增加而有所降低。七天后的信号仍保留了原值的93.3%,表明本发明制备的传感器稳定性良好。
实施例2
一种双信号无酶的信号放大RNA纳米生物传感器检测miRNA-16的应用,具体为:
将实施例1制备的Fc/MB/NEW电极放入配制好的200uL不同浓度的目标miRNA-16的溶液中。为了使目标miRNA能够更好的杂交,选择的条件为37℃恒温下杂交12h。杂交后的电极在含有5mM MgCl2和140mM NaCl的0.01M PBS缓冲溶液中浸泡处理,其SWV如图6A所示,a曲线是目标物浓度为0的空白对照,当目标miRNA浓度从100fM变化100nM时,可观察到MB 的峰电流渐渐增强,Fc的峰随之减小。如图6B显示的是亚甲基蓝与二茂铁的电流信号(扣除空白后)与目标物miRNA-16浓度的关系,发现两个峰信号的大小均和目标物浓度的对数成线性。因此将亚甲基蓝的增大值和二茂铁的减小值之和作为我们最终信号的变化值,即:
ΔI=ΔIMB+|ΔIFc|
这种方式可使制备的传感器对特定RNA的电化学响应实现信号放大。
使用“ΔIMB+ΔIFc”作为响应信号来定量测定miRNA浓度,结果如图7所示,可以观察到,“ΔIMB+ΔIFc”值也随着miRNA浓度的增加而线性增加。在RNA浓度从100fM到100nM,相应的线性关系是(ΔIMB+ΔIFc)(pA)=296.38+21.059lgCmiRNA(M)(R2=0.9916),可以看出该传感器的线性响应范围相对较宽。
实施例3
将制备的传感器用于人的血液样品环境下RNA的微量检测。将50uL的血清加入到10mL0.01M的PBS缓冲溶液中作环境溶液。取该环境溶液5mL加入5uL 5uM/L的miRNA-16标准液,将实施例1制备的Fc/MB/NEW电极与该样品孵育12小时,在含有5mM MgCl2和140mM NaCl的0.01M PBS缓冲溶液中浸泡处理,以方波伏安测定。后依次在该溶液中加入5uL 5uM/L的miRNA-16标准溶液,记录电化学信号变化。通过标准加入法在人体血清环境下检测回收率,如表1所示。
表1
从表1可以得到,回收率保持在96.0%~104.6%左右,说明本发明制备的传感器能够成功的在复杂样品环境中实现对miRNA-16的灵敏响应,具备实际样品检测的能力。
Claims (8)
1.一种双信号无酶的信号放大RNA纳米生物传感器的制备方法,其特征在于,所述制备方法包括以下步骤:
1)将探针1P-MB与DTT缓冲溶液混合均匀后,静置;然后加入乙酸溶液和乙酸钠溶液,再加入乙醇,混合,离心,除去上清液,干燥,即得处理后的探针1;
2)将步骤1)处理后的探针1用缓冲溶液溶解后,将金纳米线电极浸入探针1缓冲溶液中,孵育后,用巯基己醇封闭,得MB/NWE电极;
3)再将MB/NWE电极置于探针2P-Fc缓冲溶液中,杂交,探针2与探针1通过碱基对的互补配对,得Fc/MB/NWE电极,即为双信号无酶的信号放大RNA纳米生物传感器;
步骤1)所述探针1P-MB序列:5’-HS-SH-C6-TGG CAG CAC ATT GCC G-C6-MB-3’;
所述探针2P-Fc序列:5’-CGC CAA TAT TTA CGT GCT GCT A-C6-Fc-3’;
所述双信号无酶的信号放大RNA纳米生物传感器检测miRNA-16。
2.根据权利要求1所述的制备方法,其特征在于,步骤2)具体为:
将步骤1)处理后的探针1用0.01M的pH7.4的PBS缓冲溶液溶解,稀释到5uM;将金纳米线电极浸入探针1缓冲溶液中,恒温37℃下孵育8-12小时;然后室温下将电极浸入100uL浓度10uM的巯基己醇溶液中,封闭2小时,得MB/NWE电极。
3.根据权利要求1或2所述的制备方法,其特征在于,步骤2)中所述金纳米线电极的制备方法为:
将直径25um的2cm金微米线装入长度8cm、内径0.64mm、外径1mm毛细管中央,在P-2000激光拉制仪下加热并水平拉断,在真空状态下,加热温度400-460℃,加热四个循环,每个循环加热15-25s,冷却35-45s,使得毛细管与金属丝熔融包裹严实;然后将加热温度设置为400-460℃,拉力130-150N,速率45-55m/s,在该条件下拉断,之后使用钨丝连通导电,经过磨擦金面露出,即为金纳米盘电极,用氢氟酸刻蚀金纳米盘电极,获得金纳米线电极。
4.根据权利要求1所述的制备方法,其特征在于,步骤3)具体为:
将MB/NWE电极置于含2uM探针2P-Fc的0.01M PBS溶液中,恒温37℃下杂交8-12小时,探针2与探针1通过碱基对的互补配对,使探针1的颈环结构解开形成较稳定的双链结构阻碍亚甲基蓝的电子传递,得到Fc/MB/NWE电极。
5.根据权利要求1所述的制备方法,其特征在于,还包括步骤4):将该生物传感器与miRNA-16杂交,验证该传感器对目标RNA的特异性灵敏响应。
6.一种权利要求1-5任一项所述制备方法制备得到的双信号无酶的信号放大RNA纳米生物传感器。
7.一种权利要求1-5任一项所述制备方法制备得到的双信号无酶的信号放大RNA纳米生物传感器检测miRNA-16的应用。
8.根据权利要求7所述的应用,其特征在于,检测方法为:
将制备的双信号无酶的信号放大RNA纳米生物传感器与不同浓度的miRNA-16杂交,杂交后的电极在含有5mM MgCl2和140mM NaCl的0.01M PBS中处理后,检测SWV响应,构建杂交前后MB和Fc峰电流变化值之和与miRNA-16的浓度的对数的线性关系,实现对miRNA-16的检测。
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