CN113186253A - 一种用于检测路易体病标志物的Cas12a-DNAzyme传感器及其制备方法 - Google Patents
一种用于检测路易体病标志物的Cas12a-DNAzyme传感器及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种用于检测路易体病标志物的Cas12a‑DNAzyme传感器及其制备方法。该传感器包括以下组分:DNAzyme和Locker杂交链D‑L溶液;sub‑1和sub‑2杂交链sub1‑2溶液;发夹H溶液;含LbCas12a、crRNA 1 μM和ssDNA‑FQ的报告器溶液;和目标物溶液。该传感器通过核酸适配体与目标α‑syn寡聚体的特异性结合,将非核酸的蛋白目标转换为核酸检测,然后利用基于DNAzyme的步行器和Cas12a的结合,两次放大检测信号,实现了血清中α‑syn寡聚体的灵敏检测。
Description
技术领域
本发明属于生物传感技术领域,具体涉及一种用于检测路易体病标志物的Cas12a-DNAzyme传感器及其制备方法。
背景技术
路易体病是一组异质性疾病,包括帕金森病、帕金森病伴痴呆和痴呆伴路易体病。其病理特征主要是突触前蛋白—α-突触核蛋白(α-synuclein, α-syn)的进行性积聚,因此常被称为突触核病。研究表明α-syn寡聚体为最具致病性的形式,并且在帕金森病患者的脑脊液和血浆中能普遍检测到α-syn寡聚体含量升高。因此,监测α-syn寡聚体的含量变化情况对这些神经退行性疾病的早期诊断具有重大意义。
目前,α-syn寡聚体的临床检测主要依赖于结构和功能磁共振成像、正电子发射断层扫描、原子力显微镜、质谱、毛细管电泳以及酶联免疫吸附法等。这些分析方法通常需要昂贵的仪器设备、复杂耗时的样品预处理以及熟练的操作人员。因此,建立简便、经济的α-syn寡聚体检测方法显得尤为重要。
规律间隔成簇短回文重复序列(Clustered Regularly Interspaced ShortPalindromic Repeats, CRISPR)系统可为细菌提供适应性免疫力,以降解入侵的核酸。在CRISPR-Cas家族中,CRISPR-Cas12a是一种原核生物脱氧核糖核酸酶,可通过RNA引导编程,以靶向互补DNA序列。Cas12a-crRNA复合物在与目标DNA结合后,Cas12a相继以顺位裂解了双链DNA的非目标链和与crRNA杂交的目标链,产生了交错的双链DNA断裂,此过程称之为Cas12a诱导的靶标的顺式切割;此外,Cas12a-crRNA与靶DNA结合形成三元复合体后,还诱导非靶单链DNA的反式切割。Cas12a的这一独特性质已被应用于核酸的检测,并为提高基于核酸诊断应用的特异性、敏感性和速度提供了一种策略。虽然Cas12a在核酸检测领域有了较多应用,但其在蛋白等非核酸目标方面的应用较少报道,有待开拓。
发明内容
为解决上述问题,本发明提供一种用于检测路易体病标志物的Cas12a-DNAzyme传感器及其制备方法。通过将Cas12a与基于DNAzyme的步行器结合,利用适配体对目标物的特异性识别,实现血清中的路易体病标志物α-syn寡聚体的灵敏检测。
为实现上述目的,本发明采用如下技术方案:
一种用于检测路易体病标志物的Cas12a-DNAzyme传感器,包括以下组分:DNAzyme和Locker杂交链D-L溶液;sub-1和sub-2杂交链sub1-2溶液;发夹H溶液;含LbCas12a、crRNA和ssDNA-FQ的报告器溶液;和目标物溶液。
一种用于检测路易体病标志物的Cas12a-DNAzyme传感器的制备方法,包括以下步骤:
(1)将DNAzyme和Locker分别溶于缓冲液B中得到浓度为100 μM的溶液;分别取100μM的DNAzyme和Locker的溶液各1 μL,溶于48 μL的缓冲液B中,混匀后于95 ºC加热5 min,自然冷却至室温,得到DNAzyme和Locker的杂交链D-L溶液,称为混合物1;
(2)将α-syn寡聚体溶于缓冲液A中制备成目标物溶液;向混合物1加入目标物溶液,于37 ºC反应2 h,得到混合物2;
(3)将sub-1和sub-2分别溶于缓冲液B中得到浓度为100 μM的溶液;将sub-1溶液和sub-2溶液分别于95 ºC加热5 min,自然冷却至室温;然后取退火后的100 μM的sub-1溶液和sub-2溶液各1 μL,溶于48 μL的缓冲液B中,混匀后在室温反应2 h,得到sub-1和sub-2的杂交链sub1-2溶液,称为混合物3;
(4)将发夹H溶于缓冲液B中得到浓度为100 μM的溶液;将2 μM的发夹H经95 ºC加热5 min并自然冷却至室温;分别取10 μL退火后的2 μM发夹H、混合物2、混合物3,混匀后于30 ºC反应2 h,得到含有目标物的混合物4;
(5)各取10 μL浓度均为500 nM的LbCas12a(购自英国New England BioLabs公司)和crRNA混匀,25 ºC反应30 min,再加入50 μL的浓度为 1 μM的ssDNA-FQ,混匀后得到报告器溶液;
(6)将70 μL的报告器溶液和30 μL的混合物4混匀后于37 ºC避光反应2 h,得到总体积为100 μL的待测溶液;在525 nm的激发光波长下测量并记录样品在540−650 nm范围内的荧光强度。
上述缓冲液A成分为:0.24% Tris-0.87% NaCl,pH = 7.4,0.2 μm滤膜过滤;缓冲液B成分为:200 mM NaCl,100 mM Tris-HCl,pH = 7.4。
上述用于组装Cas12a-DNAzyme传感器的DNA序列包括:
DNAzyme:5'-GGCGGTACCAGGTCAAAGGTGGGTGAGGGGACGCCAAGAGTCCCCGCGGTTAGATAGACCGTTCGCG-3';
Locker: 5'-GGTGGCTGGAGGGGGCGCGAACGGTCTAT-3';
Sub-1:5'-TTTGCAGTATCAGTCTATCTAT/rA/GGAAGTACCGCCGTACTGCAAACTGGGTGTGTCGTCTGGTTGGGTG-3';
Sub-2:5'-TTTGCAGTATCAGTCTATCTAT/rA/GGAAGTACCGCCGTACTGCAAACGACCAACCCAGCACCCAACCAGA-3';
发夹H:5'-ATCAGTCTATGCAATAGATAGACTGATACTGCAAA-3';
ssDNA-FQ: 5'-Cy3-TTATT-BHQ2-3';
所述crRNA序列为:5'-UAAUUUCUACUAAGUGUAGAUCAGUAUCAGUCUAUCUAU-3'。
本发明的显著优点在于:
(1)本发明利用适配体与目标物的特异性结合,将非核酸的目标转为核酸检测,扩展了Cas12a的应用范围;
(2)本发明将DNAzyme与Cas12a结合,使信号得到了两次放大,检测限更低,灵敏度更高;
(3)本发明与传统的检测方法相比,无需昂贵的仪器设备、复杂耗时的样品预处理以及熟练的操作人员,更加方便经济。
附图说明
图1是用于检测路易体病标志物的Cas12a-DNAzyme传感器检测原理示意图。
图2是实施例1中加入不同浓度的目标物后的荧光响应图。
图3是实施例2中加入不同干扰物与目标物的荧光响应图。
具体实施方式
为了使本发明所述的内容更加便于理解,下面结合具体实施方式对本发明所述的技术方案做进一步的说明,但是本发明不仅限于此。
一种用于检测路易体病标志物的Cas12a-DNAzyme传感器,包括以下组分:DNAzyme和Locker杂交链D-L溶液;sub-1和sub-2杂交链sub1-2溶液;发夹H溶液;含LbCas12a、crRNA和ssDNA-FQ的报告器溶液;和目标物溶液。
上述缓冲液A成分为:0.24% Tris-0.87% NaCl,pH = 7.4,0.2 μm滤膜过滤;缓冲液B成分为:200 mM NaCl,100 mM Tris-HCl,pH = 7.4。
上述用于组装Cas12a-DNAzyme传感器的DNA序列包括:
DNAzyme:5'-GGCGGTACCAGGTCAAAGGTGGGTGAGGGGACGCCAAGAGTCCCCGCGGTTAGATAGACCGTTCGCG-3';
Locker: 5'-GGTGGCTGGAGGGGGCGCGAACGGTCTAT-3';
Sub-1:5'-TTTGCAGTATCAGTCTATCTAT/rA/GGAAGTACCGCCGTACTGCAAACTGGGTGTGTCGTCTGGTTGGGTG-3';
Sub-2:5'-TTTGCAGTATCAGTCTATCTAT/rA/GGAAGTACCGCCGTACTGCAAACGACCAACCCAGCACCCAACCAGA-3';
发夹H: 5'-ATCAGTCTATGCAATAGATAGACTGATACTGCAAA-3';
ssDNA-FQ: 5'-Cy3-TTATT-BHQ2-3';
所述crRNA序列为:5'-UAAUUUCUACUAAGUGUAGAUCAGUAUCAGUCUAUCUAU-3'。
本发明的一种用于检测路易体病标志物的Cas12a-DNAzyme传感器检测原理示如图1所示。由于DNAzyme和DNA锁(Locker)之间的杂交,在目标不存在的情况下,DNAzyme步行器不起作用, 无法产生激活Cas12a的DNA双链H-S,未激活的Cas12a不能切割单链ssDNA-FQ。然而,在目标存在的情况下,末端含有目标适配体序列的Locker优先识别并结合目标α-syn,释放出游离的DNAzyme。紧接着DNAzyme与串联体轨道上含有底物的发夹依次结合并切割底物,激活DNAzyme步行器,得到大量的单链S,完成了检测过程的第一次放大。然后,单链S通过与发夹H的粘性末端杂交并打开H, 形成双链H-S。H-S作为Cas12a的靶DNA,Cas12a诱导双链H-S的顺式切割,从而激活Cas12a。成功激活的Cas12a紧接着又发挥其反式切割非靶单链DNA的作用,将大量的两端分别标记荧光团和淬灭团的短单链ssDNA-FQ裂解,实现了检测过程的第二次放大,从而得到显著增强的荧光信号。
实施例1 一种用于检测路易体病标志物的Cas12a-DNAzyme传感器的制备方法,包括以下步骤:
1)将DNAzyme和Locker分别溶于缓冲液B中得到浓度为100 μM的溶液;分别取100μM的DNAzyme和Locker的溶液各1 μL,溶于48 μL的缓冲液B中,混匀后于95 ºC加热5 min,自然冷却至室温,得到DNAzyme和Locker的杂交链D-L溶液,称为混合物1;
2)将α-syn寡聚体溶于缓冲液A中制备成目标物溶液,在混合物1中加入不同浓度的目标物溶液,得到含有目标物终浓度分别为0,1 fM,100 fM,10 pM,1 nM,100 nM的溶液,于37 ºC反应2 h,得到混合物2;
3)将sub-1和sub-2分别溶于缓冲液B中得到浓度为100 μM的溶液;将sub-1溶液和sub-2溶液分别于95 ºC加热5 min,自然冷却至室温;然后取退火后的100 μM的sub-1溶液和sub-2溶液各1 μL,溶于48 μL的缓冲液B中,混匀后在室温反应2 h,得到sub-1和sub-2的杂交链sub1-2溶液,称为混合物3;
4)将发夹H溶于缓冲液B中得到浓度为100 μM的溶液,将2 μM的发夹H经95 ºC加热5 min并自然冷却至室温;分别取10 μL退火后的2 μM发夹H、混合物2、混合物3,混匀后于30ºC反应2 h,得到含有目标物的混合物4;
5)各取10 μL浓度均为500 nM的LbCas12a(购自英国New England BioLabs公司)和crRNA混匀,25 ºC反应30 min。再加入50 μL终浓度为 1 μM的ssDNA-FQ,混匀后得到报告器溶液;
6)将70 μL的报告器溶液和30 μL的混合物4混匀后于37 ºC避光反应2 h,得到总体积为100 μL的待测溶液,在525 nm的激发光波长下测量并记录样品在540−650 nm范围内的荧光强度。
结果如图2所示,图2显示了Cas12a-DNAzyme体系中荧光变化与目标浓度之间的关系。图中表明,荧光强度随目标浓度(从0到100 nM)的升高而增加。同时,根据空白加3σ(标准差的3倍)方法,计算出检测限达0.44 fM。
实施例2 一种用于检测路易体病标志物的Cas12a-DNAzyme传感器的制备方法,包括以下步骤:
步骤1)同实施例1;
步骤2)选择性实验,采用血清中会与目标物α-syn共存的两种蛋白(免疫球蛋白G,IgG和人血清白蛋白,HSA)来考察Cas12a-DNAzyme传感器的选择性。在步骤1)所得杂交链D-L的混合物1中分别加入目标物α-syn、两种蛋白、混合物(α-syn和IgG),使各种蛋白的终浓度均为100 nM,于37 ºC反应2 h,得到含有不同蛋白的混合物2;
步骤3)至步骤6)同实施例1。
结果如图3所示,与其他干扰蛋白相比,目标物α-syn寡聚体引起的相对荧光信号至少有11倍的增加(587/52)。不仅如此,在含有相同浓度干扰蛋白的目标物混合物中也显示了很强的信号,这表明开发的传感平台对α-syn寡聚体具有良好的特异性。
实施例3 一种用于检测路易体病标志物的Cas12a-DNAzyme传感器的制备方法,包括以下步骤:
将人血清用含200 mM NaCl的超纯水稀释10倍代替实施例1中所用到的缓冲液B。然后按实施例1中所述步骤,使用标准加样法进行回收实验。对三种不同浓度(100 fM,1 pM和10 pM)的目标物进行回收测定。通过三个独立的实验计算相对标准偏差(RSD)。
为了研究所提出的Cas12a-DNAzyme生物传感器在复杂生物样品中的适用性,我们使用健康的人血清样品进行了回收实验,以确定Cas12a-DNAzyme传感器用于α-syn寡聚体检测的可靠性。将各种不同浓度的α-syn寡聚体加入人血清样品中。结果见表1,回收率为98.0%−101.2%,由三个独立的实验计算的RSD为3.78%−7.04%。表明Cas12a-DNAzyme生物传感器在实际样品中具有出色的α-syn寡聚体检测能力。
表1
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
SEQUENCE LISTING
<110> 福州大学
<120> 一种用于检测路易体病标志物的Cas12a-DNAzyme传感器及其制备方法
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Claims (5)
1.一种用于检测路易体病标志物的Cas12a-DNAzyme传感器,其特征在于,包括以下组分:DNAzyme和Locker杂交链D-L溶液;sub-1和sub-2杂交链sub1-2溶液;发夹H溶液;含LbCas12a、crRNA 和ssDNA-FQ的报告器溶液;和目标物溶液。
2.根据权利要求1所述的一种用于检测路易体病标志物的Cas12a-DNAzyme传感器,其特征在于:用于组装Cas12a-DNAzyme传感器的DNA序列包括以下序列:
DNAzyme:5'-GGCGGTACCAGGTCAAAGGTGGGTGAGGGGACGCCAAGAGTCCCCGCGGTTAGATAGACCGTTCGCG-3';
Locker: 5'-GGTGGCTGGAGGGGGCGCGAACGGTCTAT-3';
Sub-1:5'-TTTGCAGTATCAGTCTATCTAT/rA/GGAAGTACCGCCGTACTGCAAACTGGGTGTGTCGTCTGGTTGGGTG-3';
Sub-2:5'-TTTGCAGTATCAGTCTATCTAT/rA/GGAAGTACCGCCGTACTGCAAACGACCAACCCAGCACCCAACCAGA-3';
发夹H: 5'-ATCAGTCTATGCAATAGATAGACTGATACTGCAAA-3';
ssDNA-FQ: 5'-Cy3-TTATT-BHQ2-3';
所述crRNA序列为:5'-UAAUUUCUACUAAGUGUAGAUCAGUAUCAGUCUAUCUAU-3'。
3.如权利要求1所述一种用于检测路易体病标志物的Cas12a-DNAzyme传感器的制备方法,其特征在于,包括以下步骤:
(1)将DNAzyme和Locker分别溶于缓冲液B中得到终浓度为100μM的溶液;分别取100μM的DNAzyme和Locker的溶液各1μL,溶于48 μL的缓冲液B中,混匀后于95 ºC加热5 min,自然冷却至室温,得到DNAzyme和Locker的杂交链D-L溶液,称为混合物1;
(2)将α-syn寡聚体溶于缓冲液A中制备成目标物溶液;向混合物1加入目标物溶液,于37 ºC反应2 h,得到混合物2;
(3)将sub-1和sub-2分别溶于缓冲液B中得到浓度为100 μM的溶液;将sub-1溶液和sub-2溶液分别于95 ºC加热5 min,自然冷却至室温;然后取退火后的100 μM的sub-1溶液和sub-2溶液各1 μL,溶于48 μL的缓冲液B中,混匀后在室温反应2 h,得到sub-1和sub-2的杂交链sub1-2溶液,称为混合物3;
(4)将发夹H溶于缓冲液B中得到浓度为100 μM的溶液;将2 μM的发夹H经95 ºC加热5min并自然冷却至室温;分别取10 μL退火后的2 μM发夹H、混合物2、混合物3,混匀后于30 ºC反应2 h,得到含有目标物的混合物4;
(5)各取10 μL浓度均为500 nM的LbCas12a和crRNA混匀,25 ºC反应30 min,再加入50μL的浓度为1 μM的ssDNA-FQ,混匀后得到报告器溶液;
(6)将70 μL的报告器溶液和30 μL的混合物4混匀后于37 ºC避光反应2 h,得到总体积为100 μL的待测溶液;在525 nm的激发光波长下测量并记录样品在540−650 nm范围内的荧光强度。
4.根据权利要求3所述的一种用于检测路易体病标志物的Cas12a-DNAzyme传感器的制备方法,其特征在于,所述缓冲液A成分为:0.24% Tris-0.87% NaCl,pH = 7.4,0.2 μm滤膜过滤;缓冲液B成分为:200 mM NaCl,100 mM Tris-HCl,pH = 7.4。
5.如权利要求1所述一种用于检测路易体病标志物的Cas12a-DNAzyme传感器在检测路易体病中的应用。
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