CN115975206B - 一种杂化mof材料及其制备和应用 - Google Patents
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Abstract
本发明属于纳米材料和生物医学领域,具体涉及一种杂化MOF材料及其制备和应用。杂化MOF材料为以带负电的Ag3AuS2@Fe3O4作为核,以双链DNA固定的ATP敏感的ZIF‑90作为壳,壳包裹核形成壳核结构的杂化MOF材料。本发明制备的杂化MOF材料具有特异性识别ADP和ATP的性能,可实现同一体系内ADP和ATP的同时检测。本发明制备的杂化MOF具有良好的稳定性、生物相容性,在临床和环境分析方面具有广阔的应用前景。
Description
技术领域
本发明属于纳米材料和生物医学领域,具体涉及一种杂化MOF材料及其制备和应用。
背景技术
二磷酸腺苷(ADP)与激酶酶活性相关,涉及许多细胞过程,包括细胞生长、***和程序性细胞死亡。三磷酸腺苷(ATP)是生命***中的主要能量货币,是早期诊断肾病、糖尿病、癌症、视网膜退行性疾病和阿尔茨海默病的生物标志物。ATP通过水解生成ADP为所有活细胞的正常运作提供大量的能量,ATP和ADP的交换反应是根据细胞的需要动态变化的。因此,ADP和ATP之间的比例是细胞能量状态变化的敏感指示器。此外,ADP/ATP比值在调节细胞信号活动中也很重要,例如,AMP活化的蛋白激酶通过感知ADP/ATP比值的变化,激活分解代谢通路或下调合成代谢通路,从而调节能量状态。
在一些研究中已经探索了测量不同细胞状态下ADP和ATP比值的方法。然而,这些荧光传感器只能提供ADP/ATP比值的信息,而不能提供核苷酸的绝对浓度,这限制了其在临床和生理分析中的应用。显然,在一个***中同时鉴别检测ADP和ATP可以为研究细胞能量状态和调节信号活动提供广泛的应用,并且通过简单的计算可以立即得到ADP/ATP比值。到目前为止,同时检测ADP和ATP的工作非常有限。因此,开发同时鉴别ADP和ATP的高灵敏度***是临床和检测领域的迫切需要。
发明内容
本发明的目的在于针对目前现有的不能在同一体系内同时检测ADP和ATP的问题,提供一种杂化MOF材料及其制备和应用。
为实现上述目的,本发明采用的技术方案为:
一种杂化MOF材料,杂化MOF材料为以带负电的Ag3AuS2@Fe3O4作为核,以双链DNA固定的ATP敏感的ZIF-90作为壳,壳包裹核形成壳核结构的杂化MOF材料。
所述壳为ATP敏感的ZIF-90,再通过酰胺反应将ADP适配体和DNA激活链组成的双链DNA固定的ATP敏感的ZIF-90纳米载体表面,进而形成杂化MOF材料的壳。
一种杂化MOF材料的制备方法,以带负电的Ag3AuS2@Fe3O4作为核,其外部包裹ATP敏感的ZIF-90,再通过酰胺反应将ADP适配体和DNA激活链组成的双链DNA固定在ZIF-90纳米载体表面,形成壳核结构的杂化MOF材料
所述杂化MOF材料的Ag3AuS2@Fe3O4内核为四氯金酸,硝酸银,四氧化三铁,硫粉按重量比为1:2:1:1加热反应生成。
所述Ag3AuS2@Fe3O4为四氯金酸,硝酸银,四氧化三铁混合在油酸体系中,加热(50-55℃)反应20-21小时,而后加入硫粉继续反应2-3小时。
所述ATP敏感的ZIF-90外壳为以Ag3AuS2@Fe3O4,2-甲醛咪唑,醋酸锌按重量比为5:1:1混合在DMF体系中,常温反应生成。
所述ATP敏感的ZIF-90外壳将Ag3AuS2@Fe3O4和2-甲醛咪唑混合在DMF中,常温(25-28℃)反应10-15分钟,而后加入醋酸锌常温(25-28℃)反应10-15分钟。
所述ADP适配体,DNA激活链和ATP敏感的ZIF-90按摩尔比4:4:3-4:4:2的比例混合。
所述ADP适配体为GGGACGGAGACGAGGGGACGAAAGUCCCCGGACAAUCAGACACGGUCUCCGUCCC;进一步的说,所述适配体可进一步被修饰;
DNA激活链为TCGTCCCAAACACACAAG GGAC。
一种杂化MOF材料的应用,所述材料可实现同一体系内ADP和ATP的同时检测。
所述材料在临床和环境分析中的应用。
本发明以杂化MOF材料为以带负电的Ag3AuS2@Fe3O4作为核,其外部包裹ATP敏感的ZIF-90,再通过酰胺反应将ADP适配体和DNA激活链组成的双链DNA固定在ZIF-90纳米载体表面,形成壳核结构的杂化MOF材料。本发明制备的杂化MOF材料具有特异性识别ADP和ATP的性能,可实现同一体系内ADP和ATP的同时检测。本发明制备的杂化MOF具有良好的稳定性、生物相容性,在临床和环境分析方面具有广阔的应用前景。
本发明的有益效果在于:
本发明以四氯金酸,硝酸银,四氧化三铁,硫粉反应生成的Ag3AuS2@Fe3O4为内核,包裹在ATP敏感的ZIF-90外壳中,再通过酰胺反应将ADP适配体和DNA激活链组成的双链DNA固定在ZIF-90纳米载体表面,形成壳核结构的杂化MOF材料,发现制备的杂化MOF材料具有特异性识别ADP和ATP的性能,可实现同一体系内ADP和ATP的同时检测,且具有良好的稳定性,选择性,不易被复杂环境干扰。在在临床和环境分析方面具有广阔的应用前景。
附图说明
图1为本发明实施例1提供的杂化MOF的TEM图,其中,A为Ag3AuS2@Fe3O4、B为ZIF-90@Ag3AuS2@Fe3O4、C为双链DNA-ZIF-90@Ag3AuS2@Fe3O4;
图2为本发明实施例2提供的杂化MOF检测ADP和ATP的荧光响应图,其中,A为杂化MOF在ADP溶液中反应30分钟、B为杂化MOF在ATP溶液中反应30分钟;
图3为本发明实施例3提供的杂化MOF在ADP和ATP溶液中反应30分钟的荧光变化图;
图4为本发明实施例4提供的杂化MOF在血清样品中检测ADP和ATP的结果。
具体实施方式
以下结合实例对本发明的具体实施方式做进一步说明,应当指出的是,此处所描述的具体实施方式只是为了说明和解释本发明,并不局限于本发明。
本发明通过形成特定的壳核结构的纳米杂化MOF,该纳米载体具有有特异性识别ADP和ATP的性能,可实现同一体系内ADP和ATP的同时检测。
实施例1:
杂化MOF的制备:
1)将20mg HAuCl4·4H2O、40mg AgNO3和20mg Fe3O4加入30mL油酸溶液中。将体系加热至55℃反应20h,然后在搅拌下加入10mL含25mg硫粉的油酸。搅拌2h后,用无水乙醇洗涤三次,再分散于5mL PBS(10mM,pH 7.4)中备用,得到Ag3AuS2@Fe3O4。(参见图1A)。
2)将48.05mg 2-咪唑甲醛(0.05M)和100μL Ag3AuS2@Fe3O4(10mg mL-1)溶于10.0mLDMF中,搅拌15min,然后缓慢加入5.0mL含0.05MZn(CH3COO)2的DMF。最终溶液连续搅拌15min,然后用DMF、超纯水和乙醇洗涤分散体,离心(10000rpm,5min)。最后,将产物分散在超纯水中,得到ZIF-90@Ag3AuS2@Fe3O4原液(10mg mL-1)。(参见图1B)。
3)将250μL ADP适配体-1(100μM,NH2-GGGACGGAGACGAGGGGACGAAAGUCCCCGGACAAUCAGACACGGUCUCCGUCCCUUGUGU)与125μL EDC(5mg mL-1)和125μL NHS(5mg mL-1)混合反应20min,加入5mg ZIF-90@Ag3AuS2@Fe3O4搅拌15h,然后将得到的DNA适配体-ZIF-90@Ag3AuS2@Fe3O4,用PBS(10mM,pH=7.4)洗涤3次,分散到500μL PBS溶液中。将20μL DNA激活链(10μM,TCGTCCCAAACACACAAGGGAC)溶液加入100μL DNA适配体-ZIF-90@Ag3AuS2@Fe3O4溶液中反应3h,PBS洗涤3次后分散到100μLPBS溶液中,再加入20μL ADP适配体-1(10μM,GUUUGGGACGGAGACGAGGGGACGAAAGUCCCCGGACAAUCAGACACGGUCUCCGUCCC)溶液,形成双链DNA-ZIF-90@Ag3AuS2@Fe3O4。(参见图1C)。
通过透射电子显微镜(TEM)表征制备的双链Ag3AuS2@Fe3O4、ZIF-90@Ag3AuS2@Fe3O4和双链DNA-ZIF-90@Ag3AuS2@Fe3O4的形貌。如图1A、图1B和图1C所示,Ag3AuS2@Fe3O4、ZIF-90@Ag3AuS2@Fe3O4和双链DNA-ZIF-90@Ag3AuS2@Fe3O4的都是均匀的纳米颗粒,平均直径分别为70.4、213.4和229.3nm。
实施例2:
杂化MOF的制备:
1)将20mg HAuCl4·4H2O、40mg AgNO3和20mg Fe3O4加入30mL油酸溶液中。将体系加热至50℃反应21h,然后在搅拌下加入10mL含25mg硫粉的油酸。搅拌3h后,用无水乙醇洗涤三次,再分散于5mL PBS(10mM,pH 7.4)中备用,得到Ag3AuS2@Fe3O4。(参见图1D)。
2)将48.05mg 2-咪唑甲醛(0.05M)和100μL Ag3AuS2@Fe3O4(10mg mL-1)溶于10.0mLDMF中,搅拌30min,然后缓慢加入5.0mL含0.05MZn(CH3COO)2的DMF。最终溶液连续搅拌30min,然后用DMF、超纯水和乙醇洗涤分散体,离心(10000rpm,5min)。最后,将产物分散在超纯水中,得到ZIF-90@Ag3AuS2@Fe3O4原液(10mg mL-1)。(参见图1E)。
3)将250μL ADP适配体-1(100μM,NH2-GGGACGGAGACGAGGGGACGAAAGUCCCCGGACAAUCAGACACGGUCUCCGUCCCUUGUGU)与125μL EDC(5mg mL-1)和125μL NHS(5mg mL-1)混合反应20min,加入10mg ZIF-90@Ag3AuS2@Fe3O4搅拌15h,然后将得到的DNA适配体-ZIF-90@Ag3AuS2@Fe3O4,用PBS(10mM,pH=7.4)洗涤3次,分散到500μL PBS溶液中。将20μL DNA激活链(10μM,TCGTCCCAAACACACAAGGGAC)溶液加入100μL DNA适配体-ZIF-90@Ag3AuS2@Fe3O4溶液中反应3h,PBS洗涤3次后分散到100μLPBS溶液中,再加入20μL ADP适配体-1(10μM,GUUUGGGACGGAGACGAGGGGACGAA AGUCCCCGGACAAUCAGACACGGUCUCCGUCCC)溶液,形成双链DNA-ZIF-90@Ag3AuS2@Fe3O4。(参见图1F)。
应用例1:
杂化MOF的刺激响应性:
1)将20μL Cas12a(10nM)和20μL crRNA(UAAUUUCUACUAAGUGUAGAUGUCCCUUGUGUGUUUGGGAC)在37℃下混合30min,再加入50nM的ssDNA(FAM-AACCGCTTCCCCGACTTCC-BHQ)20μL,制备含非活性Cas12a和荧光DNA的报告液。报告液现配现用。在20μL含5nM浓度ADP的PBS缓冲液(10mM,pH=7.4)中加入20μL实施例1制备所得DNA-ZIF-90@Ag3AuS2@Fe3O4(1mg mL-1)溶液检测ADP,孵育5min后,加入60μL报告液,37℃孵育25min,60℃加热5min停止酶促反应,并测定溶液的荧光。(参见图2A)。
2)在20μL含5nM浓度ATP的PBS缓冲液(10mM,pH=7.4)中加入20μL实施例1制备的双链DNA-ZIF-90@Ag3AuS2@Fe3O4(1mg mL-1)溶液检测ATP。孵育5min后,加入60μL PEI-CDs溶液(1mg mL-1),孵育15min,测定溶液的荧光。(参见图2B)。
如图2A所示,双链DNA-ZIF-90@Ag3AuS2@Fe3O4的加入并没有导致荧光响应的显著增加,然而,在ADP的作用下,双链DNA-ZIF-90@Ag3AuS2@Fe3O4可以释放出激活链,产生显著的荧光信号。如图2B所示,从图3D中可以看出,PEI-CDs具有较高的荧光,当引入双链DNA-ZIF-90@Ag3AuS2@Fe3O4和ATP后荧光显著下降,而在没有ATP的情况下引入双链DNA-ZIF-90@Ag3AuS2@Fe3O4几乎不会导致荧光猝灭。
应用例2:
杂化MOF对ADP和ATP同时检测:
为同时检测ADP和ATP,在含ADP和ATP的20μL PBS缓冲液(10mM,pH=7.4,缓冲溶液中ADP和ATP的浓度分别5nM)中加入40μL双链DNA-ZIF-90@Ag3AuS2@Fe3O4和ATP(1mg mL-1)溶液。孵育5min后,加入120μL报告溶液和20μL PEI-CDs溶液(6mg mL-1),37℃孵育25min,60℃加热5min,测定荧光响应。
图3A-D为探测***对无靶标、单靶标和双靶标的荧光光谱。与没有ADP和ATP的情况下产生的荧光强度相比(图3A),ADP的存在使520nm处的荧光发射从1174.2a.u增加到8562.4a.u,而450nm处的荧光信号变化不大(图3B)。同样,ATP的加入导致450nm处的荧光信号从10343.6a.u显著下降到1903.7a.u.,而520nm处的荧光发射没有明显的信号变化(图3C)。同时加入ADP和ATP,导致ssDNA报告基因520nm处的荧光发射显著增加,PEI-CDs 450nm处的荧光发射显著降低(图3D)。
应用例3:
杂化MOF对生物样品中ADP和ATP同时检测:
为了同时检测实际样品中的ADP和ATP,采用人血清样品进行回收实验。在PBS(10mM,pH=7.4)稀释的10%人血清中加入一系列浓度的ADP和ATP标准品,通过荧光反应测定溶液中ADP和ATP的浓度。如图4所示,ADP的加标回收率分别为95.8、102.8、104.7、106.9%,RSD在2.8%~4.6%之间。ATP检测的加标回收率分别为107.2、97.7、105.8、106.3%,RSD在2.4~4.8%之间。这些结果表明,所提出的生物传感器具有良好的实用性,可用于实际样品中ADP和ATP的超灵敏定量。
Claims (10)
1.一种杂化MOF材料,其特征在于:杂化MOF材料为以带负电的Ag3AuS2@Fe3O4作为核,以双链DNA固定的ATP敏感的ZIF-90作为壳,壳包裹核形成壳核结构的杂化MOF材料。
2.按权利要求1所述的杂化MOF材料,其特征在于:所述壳为ATP敏感的ZIF-90,再通过酰胺反应将ADP适配体和DNA激活链组成的双链DNA固定的ATP敏感的ZIF-90纳米载体表面,进而形成杂化MOF材料的壳。
3.一种权利要求1所述的杂化MOF材料的制备方法,其特征在于:以带负电的Ag3AuS2@Fe3O4作为核,其外部包裹ATP敏感的ZIF-90,再通过酰胺反应将ADP适配体和DNA激活链组成的双链DNA固定在ZIF-90纳米载体表面,形成壳核结构的杂化MOF材料。
4.按权利要求3所述的杂化MOF材料的制备方法,其特征在于:所述杂化MOF材料的Ag3AuS2@Fe3O4内核为四氯金酸,硝酸银,四氧化三铁,硫粉按重量比为1:2:1:1加热反应生成。
5.按权利要求4所述的杂化MOF材料的制备方法,其特征在于:所述Ag3AuS2@Fe3O4为四氯金酸,硝酸银,四氧化三铁混合在油酸体系中,加热至50-55℃反应20-21小时,而后加入硫粉继续反应2-3小时。
6.按权利要求3或4所述的杂化MOF材料的制备方法,其特征在于:所述ATP敏感的ZIF-90外壳为以Ag3AuS2@Fe3O4,2-甲醛咪唑,醋酸锌按重量比为5:1:1混合在DMF体系中,常温反应生成。
7.按权利要求6所述的杂化MOF材料的制备方法,其特征在于:所述ATP敏感的ZIF-90外壳为将Ag3AuS2@Fe3O4和2-甲醛咪唑混合在DMF中,常温25-28℃反应10-15分钟,而后加入醋酸锌,常温25-28℃反应10-15分钟,生成。
8.按权利要求7所述的杂化MOF材料的制备方法,其特征在于:所述ADP适配体,DNA激活链和ATP敏感的ZIF-90按摩尔比4:4:3-4:4:2的比例混合。
9. 按权利要求8所述的杂化MOF材料的制备方法,其特征在于:所述ADP适配体为GGGACGGAGACGAGGGGACGAAAGUCCCCGGACAAUCAGA CACGGUCUCCGUCCC;DNA激活链为TCGTCCCAAACACACAAG GGAC。
10.一种权利要求1所述杂化MOF材料的非疾病诊断与治疗的应用,其特征在于:所述材料可实现同一体系内ADP和ATP的同时检测。
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