CN113376132A - 一种基于介孔硫化铜复合材料、制备方法及检测方法 - Google Patents
一种基于介孔硫化铜复合材料、制备方法及检测方法 Download PDFInfo
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Abstract
本发明属于生物医药技术领域,公开了一种基于介孔硫化铜复合材料、制备方法及检测方法,制备复合材料,将DOX负载在HCuS孔道内,MnO2NS封堵DOX,通过释放DOX的量来检测GSH;将Mn2+作为DNAzyme的金属辅助因子对DNAzyme结构进行切割,结合CHA反应,通过荧光信号检测miRNA‑21。本发明通过MnO2NS与谷胱甘肽反应并生成Mn2+,MnO2NS分解释放DOX,DOX的释放量与GSH的含量成正比;通过静电吸附作用吸附在MnO2NS上的发卡DNA,在miRNA‑21存在的情况下引发催化发夹组装循环反应,实现对miRNA‑21的检测;本发明制备的复合材料实现双组分肿瘤标志物同时检测。
Description
技术领域
本发明属于生物医药技术领域,尤其涉及一种基于介孔硫化铜复合材料、制备方法及检测方法。
背景技术
目前,中空介孔硫化铜(HCuS)的内部空腔较大,具有丰富的介孔结构。HCuS的独特纳米结构使其成为潜在的药物载体。Li等人将两种功能性抗肿瘤药物阿霉素(DOX)和二氢卟酚(Ce6)装入HCuS,又通过PCM封堵孔道防止药物泄露,形成纳米复合物H-CuS@PCM/DOX/Ce6(HPDC NP)。Deng等通过在UCNP上原位还原了UCNP/MnO2 NS组件,基于镧系元素掺杂的上转换纳米颗粒和二氧化锰纳米片相结合,可快速、选择性地检测水溶液和活细胞中的谷胱甘肽。
具有宽吸收光谱的MnO2 NS与荧光纳米材料的荧光激发/发射光谱重叠,因此具有很强的荧光猝灭能力,这使其在基于荧光共振能量转移(FRET)的荧光传感中具有广阔的前景。MnO2 NS已广泛用作荧光受体,与基于FRET的荧光供体探针构成能量供体-受体对。在还原性物质(如GSH)的存在下,具有强氧化性和催化能力的MnO2 NS可以快速分解,从而导致释放载药或可用于检测生物样品中的还原性物质。
谷胱甘肽(GSH)通过在体内氧化和还原状态之间的转换在调节生物体的氧化还原平衡中起重要作用。GSH在人体血液中含量与人体老化、老年痴呆、癌等疾病密切相关。癌细胞中GSH的浓度高达0.5-10mM,是正常细胞的四倍。因此GSH作为一种肿瘤标志物,其检测灵敏度与癌症早期诊断密切相关。
miRNA与多种疾病(例如癌症,糖尿病)密切相关,因此引起了人们的广泛关注。然而,细胞内miRNA的丰度始终低于阈值,无法实现准确的分析。近年来,己经开发了基于动态DNA自组装的无酶核酸扩增技术,用于检测靶标,以及活细胞中RNA的成像,包括催化发夹组装(CHA)、滚环扩增(RCA)、杂交链式反应(HCR)、聚合酶链反应(PCR)等。
CHA是一种焓驱动且无酶的扩增技术,可用于简单地检测核酸,并在活细胞中得到了应用。与HCR相似,CHA使用两种亚稳态的发夹存储势能。发夹彼此互补。然而,互补结构域被锁定在单个发夹结构内,并且当不存在靶标时,它们的反应被动力学捕获。靶核酸(例如待检测的miRNA)与一个发夹的脚尖区域杂交,从而将其打开并允许第二个发夹与其杂交。结果是形成三个寡核苷酸的半稳定三元复合物。然后将miRNA置换并可以引发另一个反应。靶链的再循环允许在几个小时内放大约100倍的信号。
DNAzyme具有高稳定性和优异的活性,在环境监测,食品监管,生物传感和基因治疗等许多领域都具有广阔的前景。特别地,DNAzyme用作信号发生器和响应特定刺激的构件,已应用于各种分析物的生物传感和生物成像。
通过上述分析,现有技术存在的问题及缺陷为:
(1)miRNA与多种疾病(例如癌症,糖尿病)密切相关,miRNA含量低无法实现准确的分析。
(2)双组分肿瘤标志物浓度相差大时,同时检测的信号易相互覆盖与遮蔽。
(3)双组分同时检测产生不同信号,难以准确定量。
(4)介孔复合探针的稳定性。
解决以上问题及缺陷的难度为:
(1)痕量miRNA检测的灵敏度。
(2)双组分同时检测准确定量存在一定的挑战。
(3)介孔硫化铜的合成及原位生长MnO2纳米片的粒径控制,表面吸附DNA探针的稳定性。
解决以上问题及缺陷的意义为:
(1)信号扩增策略对于对低含量肿瘤标志物的超敏感检测至关重要。
(2)功能化复合探针用于双组分同时检测可以缩短检测时间,降低样品消耗及检测成本。
发明内容
针对现有技术存在的问题,本发明提供了一种基于介孔硫化铜复合材料、制备方法及检测方法。
本发明是这样实现的,一种基于介孔硫化铜的复合材料制备方法,包括:
通过模板法合成介孔硫化铜纳米粒子(HCuS),将DOX吸附在HCuS的孔道内,并在HCuS表面原位合成二氧化锰纳米片(MnO2 NS)。
进一步,所述通过模板法合成介孔硫化铜纳米粒子(HCuS)包括:
(1)HCuS的合成:
在磁力搅拌下,将CuCl2·2H2O溶液加入到含有PVP-K30的去离子水中;然后加入氢氧化钠溶液;10min后,缓慢加入2.5μL的水合肼生成橙黄色过氧化铜溶液;反应5min后,在上述溶液中加入Na2S·9H2O溶液,将反应液立刻转移至60℃水浴锅中磁力搅拌并加热2h,溶液冷却至室温后,11000r/min离心15min,用去离子水洗涤上述溶液,反复进行三次;
(2)DOX-HCuS@MnO2纳米粒子的合成:
将不同浓度的DOX溶液100μL和HCuS添加到PBS中;然后将混合物在室温反应6小时;悬浮液通过11000rpm离心15分钟,并用PBS洗涤3次去除游离DOX,生成DOX-HCuS溶液。将DOX-HCuS溶液分散在MES缓冲溶液中;再在超声处理下将KMnO4溶液加入反应溶液中;超声30min以产生DOX-HCuS@MnO2纳米粒子。
进一步,所述在HCuS表面原位合成二氧化锰纳米片(MnO2 NS)包括:
所有DNA探针首先溶解在PBS缓冲液中;将终浓度1×10-5M的DNAzyme1、DNAzyme 2和底物链混合,然后在37℃反应12h;将10μL上述混合液与H1、H2混合后加入至二氧化锰纳米片溶液100μL并混合10min,然后加入结合缓冲液,该混合物在室温下再孵育20min。
本发明的另一目的在于提供一种基于介孔硫化铜复合材料,所述基于介孔硫化铜复合材料利用所述基于介孔硫化铜的复合材料制备方法制备。
本发明的另一目的在于提供一种双组分肿瘤标准物同时检测的方法,所述双组分肿瘤标准物同时检测的方法包括:
首先制备复合材料,将DOX负载在HCuS孔道内,MnO2 NS封堵DOX,通过释放DOX的量来检测GSH;将Mn2+作为DNA zyme的金属辅助因子对DNAzyme结构进行切割,结合CHA反应,通过荧光信号检测miRNA-21。
进一步,所述双组分肿瘤标准物同时检测的方法包括以下步骤:
步骤一,进行HCuS的合成;
步骤二,进行DOX-HCuS@MnO2纳米粒子的合成;
步骤三,进行DNA在MnO2 NS上的负载;
步骤四,荧光检测GSH;
步骤五,荧光检测miRNA-21。
进一步,步骤一中,所述HCuS的合成,包括:
在磁力搅拌下,将50μL,0.5M的CuCl2·2H2O溶液加入到含有0.12g PVP-K30的12.5mL去离子水中;加入12.5mL氢氧化钠溶液,pH 9.0;10min后,缓慢加入2.5μL的水合肼生成橙黄色过氧化铜溶液;
反应5min后,在溶液中加入100μL 320mg/mL的Na2S·9H2O溶液,将反应液立刻转移至60℃水浴锅中磁力搅拌并加热2h;溶液冷却至室温后,11000r/min离心15min,用去离子水洗涤所述溶液,反复进行三次。
进一步,步骤二中,所述DOX-HCuS@MnO2纳米粒子的合成,包括:
将不同浓度的DOX溶液100μL和HCuS 100μL添加到pH 7.4,10mM,800μL的PBS中,将所述混合物在室温反应6h;悬浮液通过11000rpm离心15min,并用PBS洗涤3次去除游离DOX,生成DOX-HCuS溶液;
将DOX-HCuS溶液分散在pH 6.0,10mM的MES缓冲溶液中;在超声处理下将10mM,100μL的KMnO4溶液加入反应溶液中;超声30min,产生DOX-HCuS@MnO2纳米粒子。
进一步,步骤三中,所述DNA在MnO2 NS上的负载,包括:
所有DNA探针首先溶解在10mM,pH为7.4的PBS缓冲液中;将终浓度1×10-5M的DNAzyme 1、DNAzyme 2和底物链混合,在37℃反应12h;
将10μL所述混合液与H1、H2混合后加入至DOX-HCuS@MnO2纳米粒子溶液100μL并混合10min,加入结合缓冲液,将混合物在室温下孵育20min。
进一步,所述结合缓冲液20mM PBS,内含150mM NaCl,pH=7.4。
进一步,所述DNAzyme 1的DNA序列如SEQ ID NO:1所示,所述DNAzyme 2的DNA序列如SEQ ID NO:2所示。
进一步,步骤四中,所述荧光检测GSH,包括:
将DOX-HCuS@MnO2分散到含有不同浓度GSH的PBS溶液中,震荡反应5h,离心取上清液,使用荧光分光光度计在594nm处测定阿霉素的荧光强度,对谷胱甘肽浓度进行定量;在GSH浓度从0到2mM之间时,DOX-HCuS@MnO2在488nm处的荧光信号逐渐增强。
进一步,步骤五中,所述荧光检测miRNA-21,包括:
通过CHA信号增强策略来对miRNA-21进行检测;
当miRNA-21存在时,miRNA-21、DNAzyme 1、DNAzyme 2、底物之间形成工字型结构;随着Mn2+的加入,DNAzyme被激活,将底物切割成两部分,形成许多的短链S1,释放的S1随后打开H1,H2又通过竞争作用与H1结合,H2的发夹结构被打开,S1重新被释放;通过测定H2荧光信号的强弱,对miRNA-21进行定量。
将最终浓度1×10-5M的DNAzyme 1、DNAzyme 2和底物链混合在杂交缓冲液中,在37℃反应12h;将混合液、H1、H2各取10μL混合,加入10μL 500μM的Mn2+,将不同浓度的DNA-21分别加入到所述溶液中,并用PBS定容至100μL,在37℃水浴反应4h,测定FAM的荧光强度;荧光强度与目标miRNA-21浓度存在正比关系。
进一步,所述miRNA-21的核苷酸序列如SEQ ID NO:3所示,所述DNA-21的核苷酸序列如SEQ ID NO:4所示,Substrate的核苷酸序列如SEQ ID NO:5所示,所述H1的核苷酸序列如SEQ ID NO:6所示,所述H2的核苷酸序列如SEQ ID NO:7所示,miRNA-141的核苷酸序列如SEQ ID NO:8所示。
结合上述的所有技术方案,本发明所具备的优点及积极效果为:为实现双组分同时检测,本发明制备了基于介孔硫化铜的纳米复合材料,通过模板法合成了介孔硫化铜纳米粒子(HCuS),将DOX吸附在HCuS的孔道内,并在HCuS表面原位合成二氧化锰纳米片(MnO2NS)。本发明可以实现双组分肿瘤标志物检测,MnO2 NS可与谷胱甘肽(GSH)反应并生成Mn2+,MnO2NS的分解可以释放DOX,DOX的释放量与GSH的含量成正比,因此可实现对GSH的检测。同时,本发明通过静电吸附作用吸附在MnO2 NS上的发卡DNA,在miRNA-21存在的情况下引发催化发夹组装循环反应。因此,本发明可以实现对miRNA-21的检测。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对本发明实施例中所需要使用的附图做简单的介绍,显而易见地,下面所描述的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下还可以根据这些附图获得其他的附图。
图1是本发明实施例提供的双组分肿瘤标准物同时检测的方法流程图。
图2是本发明实施例提供的基于阿霉素-介孔硫化铜@二氧化锰-DNAzyme复合材料对双组分肿瘤标志物检测原理图。
图3是本发明实施例提供的DOX荧光信号随GSH浓度变化示意图。
图4(a)-图4(d)是本发明实施例提供的FAM荧光信号随目标miRNA-21浓度变化示意图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
针对现有技术存在的问题,本发明提供了一种双组分肿瘤标准物同时检测的方法,下面结合附图对本发明作详细的描述。
本发明提供一种基于介孔硫化铜的复合材料制备方法,包括:
通过模板法合成介孔硫化铜纳米粒子(HCuS),将DOX吸附在HCuS的孔道内,并在HCuS表面原位合成二氧化锰纳米片(MnO2 NS)。
本发明还提供一种基于介孔硫化铜复合材料,所述基于介孔硫化铜复合材料利用所述基于介孔硫化铜的复合材料制备方法制备。
如图1所示,本发明实施例提供的双组分肿瘤标准物同时检测的方法包括以下步骤:
S101,进行HCuS的合成;
S102,进行DOX-HCuS@MnO2纳米粒子的合成;
S103,进行DNA在MnO2 NS上的负载;
S104,荧光检测GSH;
S105,荧光检测miRNA-21。
下面结合实施例对本发明的技术方案作进一步描述。
为实现双组分肿瘤标志物同时检测,本发明制备了基于介孔硫化铜的纳米复合材料,通过模板法合成了介孔硫化铜纳米粒子(HCuS),将DOX吸附在HCuS的孔道内,并在HCuS表面原位合成二氧化锰纳米片(MnO2 NS)。本发明可以实现双组分肿瘤标志物检测,MnO2 NS可与谷胱甘肽(GSH)反应并生成Mn2+,MnO2NS的分解可以释放DOX,DOX的释放量与GSH的含量成正比,因此可实现对GSH的检测。同时,本发明通过静电吸附作用吸附在MnO2 NS上的发卡DNA,在miRNA-21存在的情况下引发催化发夹组装循环反应。因此,本发明可以实现对miRNA-21的检测。
本发明的目的是实现肿瘤标志物双组分的同时检测。首先制备复合材料,将DOX负载在HCuS孔道内,MnO2 NS封堵DOX,通过释放DOX的量来检测GSH。Mn2+可作为DNAzyme的金属辅助因子对DNAzyme结构进行切割,结合CHA反应,通过荧光信号检测miRNA-21。
本发明提供的的方法包括以下步骤:
(1)HCuS的合成
(2)DOX-HCuS@MnO2纳米粒子的合成
(3)DNA在MnO2 NS上的负载
(4)荧光检测GSH
(5)荧光检测miRNA-21
实验步骤
本发明所用到的DNA序列
DNAzyme 1 GATACCCTGGGCGTCCGAGCCTGAT AAGCTA(SEQ ID NO:1)
DNAzyme 2 TCAACATCAGTCGGTCGAAATAGTGAGTCGCTCG(SEQ ID NO:2)
miRNA-21 UAGCUUAUCAGACUGAUGUUGA(SEQ ID NO:3)
DNA-21 TAGCTTATCAGACTGATGTTGA(SEQ ID NO:4)
Substrate CGAGCGACTCACTATrAGCGCCCAGGGTATCCAGCTG(SEQ ID NO:5)
H1
GCCCAGCTGGATACCCTGGGCGCACTCGTATCCAGCTGGGCCTTGC(SEQ ID NO:6)
H2
GCCCAGCTGGATACGAGTGCGCCCAGGGTATCCAGCGCACTCGTATCC(SEQ ID NO:7)
miRNA-141 UAACACUGUCUGGUAAAGAUGG(SEQ ID NO:8)
一、制备过程
1.HCuS的合成
在磁力搅拌下,将CuCl2·2H2O溶液(50μL,0.5M)加入到含有PVP-K30(0.12g)的去离子水(12.5mL)中。然后加入氢氧化钠溶液(12.5mL,pH 9.0)。10min后,缓慢加入2.5μL的水合肼生成橙黄色过氧化铜溶液。反应5min后,在上述溶液中加入Na2S·9H2O溶液(100μL,320mg/mL),将反应液立刻转移至60℃水浴锅中磁力搅拌并加热2h,溶液冷却至室温后,11000r/min离心15min,用去离子水洗涤上述溶液,反复进行三次。
2.DOX-HCuS@MnO2纳米粒子的合成
将不同浓度的DOX溶液100μL和HCuS(100μL)添加到PBS(pH 7.4,10mM,800μL)中。然后将混合物在室温反应6小时。悬浮液通过11000rpm离心15分钟,并用PBS洗涤3次去除游离DOX,生成DOX-HCuS溶液。将DOX-HCuS溶液分散在MES缓冲溶液(pH 6.0,10mM)中。之后在超声处理下将KMnO4溶液(10mM,100μL)加入反应溶液中。超声30min以产生DOX-HCuS@MnO2纳米粒子。
3.DNA在MnO2 NS上的负载
所有DNA探针首先溶解在PBS缓冲液(10mM,pH=7.4)中。将终浓度1×10-5M的DNAzyme 1、DNAzyme 2和底物链混合,然后在37℃反应12h。将10μL上述混合液与H1、H2混合后加入至DOX-HCuS@MnO2纳米粒子溶液100μL并混合10min,然后加入结合缓冲液(20mMPBS,内含150mM NaCl,pH=7.4),该混合物在室温下再孵育20min。
二、荧光检测
1.检测GSH
将DOX-HCuS@MnO2分散到含有不同浓度GSH的PBS溶液中,震荡反应5h,离心取上清液,使用荧光分光光度计在594nm处测定阿霉素的荧光强度,对谷胱甘肽浓度进行定量。在GSH浓度从0到2mM之间时,DOX-HCuS@MnO2在488nm处的荧光信号逐渐增强。
2.检测miRNA-21
通过CHA信号增强策略来对的miRNA-21进行检测。当miRNA-21存在时,miRNA-21、DNAzyme 1、DNAzyme 2、底物之间形成了工字型结构。随着Mn2+的加入,DNAzyme被激活,它可以将底物切割成两部分。因此,可以形成许多的短链S1,释放的S1随后打开H1,H2又通过竞争作用与H1结合,H2的发夹结构被打开,S1重新被释放。通过测定H2荧光信号的强弱,可以对miRNA-21进行定量和成像。
将终浓度1×10-5M的DNAzyme 1、DNAzyme 2和底物链混合在杂交缓冲液中,然后在37℃反应12h。将上述混合液、H1、H2各取10μL混合,加入10μL的Mn2+(500μM),再将不同浓度的DNA-21分别加入到上述溶液,并用PBS定容至100μL,然后在37℃水浴反应4h,测定FAM的荧光强度。荧光强度与目标miRNA-21浓度存在正比关系。
图2是基于阿霉素-介孔硫化铜@二氧化锰-DNAzyme复合材料对双组分肿瘤标志物检测原理图,图3是DOX荧光信号随GSH浓度变化,图4是FAM荧光信号随目标miRNA-21浓度变化。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,都应涵盖在本发明的保护范围之内。
序列表
<110> 青岛科技大学
<120> 一种基于介孔硫化铜复合材料、制备方法及检测方法
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tagcttatca gactgatgtt ga 22
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<211> 46
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gcccagctgg ataccctggg cgcactcgta tccagctggg ccttgc 46
<210> 7
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gcccagctgg atacgagtgc gcccagggta tccagcgcac tcgtatcc 48
<210> 8
<211> 22
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 8
uaacacuguc ugguaaagau gg 22
Claims (10)
1.一种基于介孔硫化铜的复合材料制备方法,其特征在于,所述基于介孔硫化铜的复合材料制备方法包括:
通过模板法合成介孔硫化铜纳米粒子(HCuS),将DOX吸附在HCuS的孔道内,并在HCuS表面原位合成二氧化锰纳米片(MnO2 NS)。
2.如权利要求1所述基于介孔硫化铜的复合材料制备方法,其特征在于,所述通过模板法合成介孔硫化铜纳米粒子(HCuS)包括:
(1)HCuS的合成:
在磁力搅拌下,将CuCl2·2H2O溶液加入到含有PVP-K30的去离子水中;然后加入氢氧化钠溶液;10min后,缓慢加入2.5μL的水合肼生成橙黄色过氧化铜溶液;反应5min后,在上述溶液中加入Na2S·9H2O溶液,将反应液立刻转移至60℃水浴锅中磁力搅拌并加热2h,溶液冷却至室温后,11000r/min离心15min,用去离子水洗涤上述溶液,反复进行三次;
(2)DOX-HCuS@MnO2纳米粒子的合成:
将不同浓度的DOX溶液100μL和HCuS添加到PBS中;然后将混合物在室温反应6小时;悬浮液通过11000rpm离心15分钟,并用PBS洗涤3次去除游离DOX,生成DOX-HCuS溶液。将DOX-HCuS溶液分散在MES缓冲溶液中;再在超声处理下将KMnO4溶液加入反应溶液中;超声30min以产生DOX-HCuS@MnO2纳米粒子。
3.如权利要求1所述基于介孔硫化铜的复合材料制备方法,其特征在于,所述在HCuS表面原位合成二氧化锰纳米片(MnO2 NS)包括:
所有DNA探针首先溶解在PBS缓冲液中;将终浓度1×10-5M的DNAzyme 1、DNAzyme 2和底物链混合,然后在37℃反应12h;将10μL上述混合液与H1、H2混合后加入至二氧化锰纳米片溶液100μL并混合10min,然后加入结合缓冲液,该混合物在室温下再孵育20min。
4.一种基于介孔硫化铜复合材料,其特征在于,所述基于介孔硫化铜复合材料利用权利要求1~3任意一项所述基于介孔硫化铜的复合材料制备方法制备。
5.一种双组分肿瘤标准物同时检测的方法,其特征在于,所述双组分肿瘤标准物同时检测的方法包括:
首先制备复合材料,将DOX负载在HCuS孔道内,MnO2 NS封堵DOX,通过释放DOX的量来检测GSH;将Mn2+作为DNA zyme的金属辅助因子对DNAzyme结构进行切割,结合CHA反应,通过荧光信号检测miRNA-21。
6.如权利要求5所述的双组分肿瘤标准物同时检测的方法,其特征在于,所述双组分肿瘤标准物同时检测的方法包括以下步骤:
步骤一,进行HCuS的合成;
步骤二,进行DOX-HCuS@MnO2纳米粒子的合成;
步骤三,进行DNA在MnO2 NS上的负载;
步骤四,荧光检测GSH;
步骤五,荧光检测miRNA-21。
7.如权利要求6所述的双组分肿瘤标准物同时检测的方法,其特征在于,步骤一中,所述HCuS的合成,包括:
在磁力搅拌下,将50μL,0.5M的CuCl2·2H2O溶液加入到含有0.12g PVP-K30的12.5mL去离子水中;加入12.5mL氢氧化钠溶液,pH 9.0;10min后,缓慢加入2.5μL的水合肼生成橙黄色过氧化铜溶液;
反应5min后,在溶液中加入100μL 320mg/mL的Na2S·9H2O溶液,将反应液立刻转移至60℃水浴锅中磁力搅拌并加热2h;溶液冷却至室温后,11000r/min离心15min,用去离子水洗涤所述溶液,反复进行三次。
8.如权利要求6所述的双组分肿瘤标准物同时检测的方法,其特征在于,步骤二中,所述DOX-HCuS@MnO2纳米粒子的合成,包括:
将不同浓度的DOX溶液100μL和HCuS 100μL添加到pH 7.4,10mM,800μL的PBS中,将所述混合物在室温反应6h;悬浮液通过11000rpm离心15min,并用PBS洗涤3次去除游离DOX,生成DOX-HCuS溶液;
将DOX-HCuS溶液分散在pH 6.0,10mM的MES缓冲溶液中;在超声处理下将10mM,100μL的KMnO4溶液加入反应溶液中;超声30min,产生DOX-HCuS@MnO2纳米粒子。
9.如权利要求6所述的双组分肿瘤标准物同时检测的方法,其特征在于,步骤三中,所述DNA在MnO2 NS上的负载,包括:
所有DNA探针首先溶解在10mM,pH为7.4的PBS缓冲液中;将终浓度1×10-5M的DNAzyme1、DNAzyme 2和底物链混合,在37℃反应12h;
将10μL所述混合液与H1、H2混合后加入至DOX-HCuS@MnO2纳米粒子溶液100μL并混合10min,加入结合缓冲液,将混合物在室温下孵育20min。
10.如权利要求6所述的双组分肿瘤标准物同时检测的方法,其特征在于,所述结合缓冲液20mM PBS,内含150mM NaCl,pH=7.4;
所述DNAzyme 1的DNA序列如SEQ ID NO:1所示,所述DNAzyme 2的DNA序列如SEQ IDNO:2所示;
步骤四中,所述荧光检测GSH,包括:
将DOX-HCuS@MnO2分散到含有不同浓度GSH的PBS溶液中,震荡反应5h,离心取上清液,使用荧光分光光度计在594nm处测定阿霉素的荧光强度,对谷胱甘肽浓度进行定量;在GSH浓度从0到2mM之间时,DOX-HCuS@MnO2在488nm处的荧光信号逐渐增强;
步骤五中,所述荧光检测miRNA-21,包括:
通过CHA信号增强策略来对的miRNA-21进行检测;
当miRNA-21存在时,miRNA-21、DNAzyme 1、DNAzyme 2、底物之间形成工字型结构;随着Mn2+的加入,DNAzyme被激活,将底物切割成两部分,形成许多的短链S1,释放的S1随后打开H1,H2又通过竞争作用与H1结合,H2的发夹结构被打开,S1重新被释放;通过测定H2荧光信号的强弱,对miRNA-21进行定量;
将终浓度1×10-5M的DNAzyme 1、DNAzyme 2和底物链混合在杂交缓冲液中,在37℃反应12h;将混合液、H1、H2各取10μL混合,加入10μL 500μM的Mn2+,将不同浓度的DNA-21分别加入到所述溶液中,并用PBS定容至100μL,在37℃水浴反应4h,测定FAM的荧光强度;荧光强度与目标miRNA-21浓度存在正比关系;
所述miRNA-21的核苷酸序列如SEQ ID NO:3所示,所述DNA-21的核苷酸序列如SEQ IDNO:4所示,Substrate的核苷酸序列如SEQ ID NO:5所示,所述H1的核苷酸序列如SEQ IDNO:6所示,所述H2的核苷酸序列如SEQ ID NO:7所示,miRNA-141的核苷酸序列如SEQ IDNO:8所示。
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