CN116765387A - 一种dna纳米花原位合成金纳米团簇和锰金属有机框架荧光适配体传感器的制备方法 - Google Patents
一种dna纳米花原位合成金纳米团簇和锰金属有机框架荧光适配体传感器的制备方法 Download PDFInfo
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Abstract
本发明涉及了一种以DNA纳米花为模板原位合成金纳米团簇(AuNCs)和锰金属有机框架的制备方法。通过水热还原法在DNA纳米花上原位合成了具有高荧光信号的AuNCs。锰金属有机框架固定发夹链2,用作局部催化发夹组装的位点,放大目标信号。加入的目标物通过与适配体发夹链1结合,影响催化发夹自组装反应,并进一步影响作用于DNA纳米花的发夹链数量,通过发夹链3的3’末端富G碱基淬灭AuNCs,产生信号变化,从而实现对目标物的灵敏检测。本发明对于食品中黄曲霉毒素B1的检测表现出了高灵敏度、高准确性和高稳定性以及优异的实际应用能力。
Description
技术领域
本发明涉及食品安全检测技术领域,具体涉及一种以DNA纳米花为模板原位合成金纳米团簇(AuNCs)和锰金属有机框架(Mn-MOF)荧光适配体传感器的制备方法。
背景技术
黄曲霉毒素是一类由黄曲霉属和寄生曲霉属真菌产生的次生代谢毒素,具有强烈的毒性和致癌性。黄曲霉毒素B1(AFB1)是黄曲霉毒素中最为广泛存在和毒性最强的一种,对人体肝脏、免疫系统和生殖系统等均有不同程度的危害。黄曲霉毒素主要通过粮食、饲料和其他食品的污染而进入人类食品链,由于黄曲霉毒素具有较强的耐热性和化学稳定性,在传统的食品加工和储存过程中,难以彻底去除或降低其含量。为了保障公众的健康和食品安全,加强对食品中黄曲霉毒素的检测具有十分重大的意义。
目前,针对AFB1的检测主要采用色谱分析方法,包括液相色谱法、液相色谱串联质谱法和气相色谱串联质谱法,这些方法能够实现高灵敏度的检测。然而,这些方法的设备和技术操作要求较高,且样品处理复杂、耗时,成本较高,不适用于大规模的食品安全检测。因此建立一种简单,高效,适用于快速检测的方法对于解决食品中AFB1超标的问题是有效的途径之一。
荧光适配体传感器是一种基于适配体进行分子识别的传感器,利用荧光基团作为信号转换器,实现对于靶分子的高灵敏度和高选择性检测,具有检测速度快,操作简便,灵敏度高等优势。适配体是一种可以特异性结合目标物的分子探针,相较于抗体,适配体具有结构简单,易于合成和修饰,适应范围广,易于制备和储存等优势。同时,适配体不受目标物的种类、大小和来源的限制,可以针对包括非天然分子在内的多种目标分子进行设计和筛选。荧光适配体传感器通常是在适配体或其互补链上进行标记,通过加入目标物后引起的构象变化从而开启或淬灭荧光信号。然而适配体进行荧光标记修饰往往具有价格昂贵,且会影响特异性识别能力的缺点,基于此,本发明首次利用DNA纳米花原位合成了具有高荧光强度的AuNCs,无标记的AuNCs荧光探针在降低了成本的同时还具有较高的稳定性。
DNA纳米花是一种基于DNA纳米技术制备的高度有序的DNA自组装结构,其形态类似于花朵,具有较高的空间精度和可编程性。DNA纳米花是由环状DNA模板链通过聚合酶驱动的循环扩增反应(rolling circle replication, RCR)自发性地组装而成,其结构的形成和稳定性依赖于DNA的序列和配对规则,能够通过精确的设计实现多层次的形态和结构功能。DNA纳米花广泛应用于药物递送、分子诊断等生物医学领域,然而由于DNA纳米花本身缺乏分子识别能力,因此在食品安全检测尤其是针对小分子真菌毒素检测的应用中受到很大的限制。基于此,本发明通过精确设计环状DNA模板链的碱基序列,通过RCR扩增反应,在DNA纳米花上复制出了大量的发夹链互补序列,使其能与参与AFB1检测部分的发夹链特异性识别。本发明利用精确设计的三条发夹链结构,实现了催化发夹组装反应,提高了检测系统的灵敏度。其中发夹3(H3)与纳米花结合后,其3’端的富G碱基(6G)靠近纳米花上的AuNCs从而引起荧光信号的淬灭,无需额外的淬灭标记基团或淬灭材料的引入,降低了传感器的成本和复杂度。此外,本发明利用Mn-MOF作为催化发夹组装的位点,提高了局部反应物的浓度,进一步提高了催化反应的效率。
本发明针对现有检测技术存在的问题,提供了一种由DNA纳米花原位合成AuNCs和Mn-MOF荧光传感器的制备方法,用于准确检测食品中的AFB1,其目的是克服目前常用检测方法中操作复杂,成本较高,灵敏度较低等缺点。本方法采用原位合成的AuNCs作为荧光信号标签并用富G序列代替淬灭基团,有效地降低了传感器的制备成本,提高了检测的灵敏度。此外,引入的Mn-MOF作为催化发夹组装的位点和,提高了检测速率和准确性。本发明能够低成本,快速,高灵敏度和高准确性地实现对食品样品中AFB1的检测,有利于发明的推广应用。
发明内容
一种DNA纳米花原位合成金纳米团簇和锰金属有机框架荧光适配体传感器的制备方法,包括以下步骤:
(1)DNA纳米花的制备:将模板链(Padlock)和引物链(Primer)共孵育,在T4 DNA连接酶作用下形成环状模板,其中, Padlock本身含有合成AuNCs的互补序列(T-rich)和H3链的互补序列;将环状模板加入Phi29 DNA聚合酶体系溶液,该体系包括1X Phi29缓冲液,dNTPs和Phi29聚合酶,在30℃下孵育,通过聚合酶驱动滚动循环放大生成具有双功能的DNA纳米花。
(2)由DNA纳米花原位合成AuNCs:将步骤(1)中得到的DNA纳米花加入到柠檬酸钠缓冲液中,并加入氯金酸溶液,置于磁力搅拌器中搅拌孵育得到荧光信号标签DNA纳米花@AuNCs。
(3)Mn-MOF的制备和修饰:将二水合醋酸锰和H2TCPP置于N,N-二甲基甲酰胺(DMF)溶液中混合均匀,室温孵育后得到Mn-MOF,随后使用EDC/NHS溶液活化Mn-MOF表面羧基,与氨基修饰的发夹2(H2)链共孵育,连接后得到Mn-MOF-H2;
(4)荧光适配体传感器的构建:将含有目标物的样品制成不同浓度的待测液,加入适配体发夹链(H1),Mn-MOF-H2和发夹3(H3)链,通过催化发夹自组装反应,获得大量的Mn-MOF-H2/H3,将其与步骤(2)制得的DNA纳米花@AuNCs混合孵育后,Mn-MOF上修饰的H3链与DNA纳米花互补结合,随后通过H3链3’端的6G序列淬灭AuNCs的荧光,通过荧光仪测量荧光强度变化,建立荧光信号响应值与目标物浓度间的关系曲线;
(5)应用于食品样品检测:经步骤(4)测得未知浓度的待测食品样品的荧光信号,将检测结果代入标准曲线得到样品中AFB1浓度。
进一步限定,步骤(1)Padlock和Primer的添加量为1-10 μL,浓度为1-10 μM,所用的T4 DNA连接酶(5 U/μL)的添加量为1-5 μL;Phi29聚合酶(10 U/μL)的添加量为1-5 μL,孵育时间为12-36 h。
进一步限定,步骤(2)中柠檬酸钠缓冲液浓度为50-100 mM,四氯金酸(1 mM)添加量为5-20 μL,孵育温度为80-90℃,孵育时间为20-60 min。
进一步限定,步骤(3)中二水合醋酸锰的添加量为20-50 mg,H2TCPP的添加量为5-20 mg,DMF体积为20-60 μL。
进一步限定,步骤(1)(4)中所述的Padlock序列为5’-P-ATG ATA TGA TCG TTGTCA CTG CCT GCT TTT TTT TTT TAT GAT GG-3’或5’-P- ATG ATA TGA TCG TTG TCA CTGCCT GCT TTT TTT TTT TTT TTT TTT TTA TGA TGG-3’或5’-P-ATG ATA TGA TCG TTG TCACTG CCT GCT TTT TTT TTT TTT TTT TTT TTT TTT TAT GAT GG-3’或5’-P-ATG ATA TGATCG TTT TTT TTT TAT GAT CGT TGT CAC TGC CTG CTT TTT TTT TTT ATG ATG G -3’中的一种;所述的Primer的序列为5’-CGA TCA TAT CAT CCA TCA TAA AAA-3’;所述的H1序列为5’-GTT GGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CCA CAC TAACAT ATT CAA GGG CAC GAG ACA CAG AG-3’;所述的H2序列为5’-CTC TGT GTC TCG TGCCCT TGA ATA TGT TAG ATG ATC GTT GTC ACT GCC TGC CTA ACA TAT TCA AGC GAA GCTC-NH2-3’;所述的H3序列为5’-TTG AAT ATG TTA GGC AGG CAG TGA CAA CGA TCA TCT AACAT ATT CAA GGG CAC GAG ACA CAG AGA TGA TCG TTG TCA CTG CCT GCG GGG GG-3’。
与现有技术相比,本发明具有如下显著优点:
1.本发明的信号标签为DNA纳米花原位合成的AuNCs,与传统以生物蛋白,氨基酸,单链DNA等为模板合成的AuNCs相比,具有更高的信号强度和稳定性。
2.本发明利用精确设计的三条发夹链结构,实现了催化发夹组装反应,提高了检测系统的灵敏度。
3.本发明利用Mn-MOF作为催化发夹组装的位点,提高了局部反应物的浓度,进一步提高了催化反应的效率。
4.本发明利用发夹链自身的碱基序列(6G)作为信号开关,可以有效地淬灭AuNCs的荧光信号,无需额外淬灭材料的引入,降低了系统的复杂度与成本。
5.基于本发明的传感策略,具有灵敏度高,准确性好,高稳定性的特点,有利于发明的推广应用。
附图说明
图1为一种DNA纳米花原位合成金纳米团簇和锰金属有机框架荧光适配体传感器的制备方法及检测黄曲霉毒素B1的示意图。
图2为本发明实施例1所构建的传感器在无AFB1(点线),存在10 ng/mL AFB1(虚线),100 ng/mL AFB1(实线)时系统的荧光强度。
图3为本发明实施例1所构建的荧光适配体传感器在其他干扰毒素存在的情况下对AFB1的选择性比较图。
图4为本发明经实施例1步骤所制备的DNA纳米花扫描电镜图,其中内嵌图的比例尺为500 nm。
图5为本发明经实施例1步骤所制备的锰金属有机框架扫描电镜图。
实施方式
下面结合附图和实施例,对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明,但不用来限制本发明的范围。
实施例
一种DNA纳米花原位合成金纳米团簇和锰金属有机框架荧光适配体传感器的制备方法及协同检测黄曲霉毒素B1的应用,其实现方法如图1所示。
一种DNA纳米花原位合成金纳米团簇和锰金属有机框架荧光适配体传感器的制备方法及协同检测黄曲霉毒素B1的应用,包括以下步骤:
(1)环状模板的制备:取1 μL 的Padlcok (1 μM)和1.5 μL的Primer(1 μM)于20 μL的1X T4连接酶缓冲液中,在PCR仪中95℃退火5 min并逐渐冷却至室温,向上述体系加入2μL的T4 DNA连接酶(5 U/μL),16℃过夜孵育12 h。
(2)DNA纳米花的制备:向步骤(1)体系加入1.5 μL的Phi29 DNA聚合酶,1 μL的dNTP和3 μL的10X Phi29 缓冲液,充分混匀后于30℃下孵育24 h。
(3)由DNA纳米花原位合成AuNCs:取步骤(2)中10 μL的DNA纳米花溶液,10 μL的氯金酸溶液(1 μM),10 μL的柠檬酸钠缓冲液(100 mM,Ph=6.0),加水补充至200 μL,于磁力搅拌器中90℃孵育30 min。
(4)锰金属有机框架(Mn-MOF)的制备和修饰:取27 mg的二水合醋酸锰于30 mL的N,N-二甲基甲酰胺(DMF)中,混合均匀;另取10 mg H2TCPP于 10 mL的DMF中,混合均匀后缓慢加入到上述醋酸锰溶液中,混合均匀后在室温下孵育12 h,产物离心后用无水乙醇洗涤3次。向Mn-MOF中加入EDC/NHS溶液(50 mM),H2链(10 mM),室温下孵育2 h,得到Mn-MOF-H2。
(5)荧光适配体传感器的构建:将含有目标物的样品制成不同浓度的待测液,加入适配体链H1,Mn-MOF-H2和H3链,通过催化发夹自组装反应,获得大量的Mn-MOF-H2/H3,将其与步骤(3)制得的DNA纳米花@AuNCs混合孵育后,Mn-MOF上修饰的H3链与DNA纳米花互补结合,随后通过H3链3’端的6G序列淬灭AuNCs的荧光,通过荧光仪测量荧光强度变化,建立荧光信号响应值与目标物浓度间的关系曲线;
(6)应用于食品样品检测:经步骤(5)测得未知浓度的待测食品样品的荧光信号,将检测结果带入标准曲线得到样品中AFB1浓度。
如图2所示,为本发明实施例1所构建的传感器在无AFB1(点线),存在10 ng/mLAFB1(虚线)和100 ng/mL AFB1(实线)时传感器的荧光强度。
实施例
一种DNA纳米花原位合成金纳米团簇和锰金属有机框架荧光适配体传感器的制备方法及检测黄曲霉毒素B1的应用,包括以下步骤:
(1)为了验证所制备的基于DNA纳米花@AuNCs和Mn-MOF的荧光适配体传感器对AFB1有特异性识别,在Tris缓冲液中加入AFB1标准品,使样品中AFB1的浓度为10 ng/mL;采用50 mM的Tris缓冲液分别配置其他干扰毒素(OTA,DON,FB1,ZEN)标准品溶液,浓度均为200 ng/mL。纵坐标对应的信号强度为不同目标物与空白样品在438 nm处的信号值。按照实施例1所构建的检测体系对上述几种不同干扰毒素标准品进行检测,检测结果如图3所示,说明本发明方法对AFB1具有较高的选择性。
实施例
一种DNA纳米花原位合成金纳米团簇和锰金属有机框架荧光适配体传感器的制备方法及检测黄曲霉毒素B1的应用,包括以下步骤:
(1)实际样品处理:取0.5 g的食品加标样品(玉米粉、花生粉),加入到10 mL 甲醇/水溶液(比例7:3)中,置于摇床上震荡20分钟,随后取出置于离心机中离心10 分钟,转速10000 转/分钟,离心完成后取出上清液得到食品样品提取液。
(2)样品检测:按照实施例1的步骤测得荧光强度信号,带入标准曲线得到样品中AFB1的浓度。
(3)以花生粉为实际样品进行测定时,以10 ng/mL加入量为基准,在花生粉中分别加入基准量0.1倍,10倍的AFB1标准品。取2 μL样品溶液,按照实施例1步骤(1)-步骤(5)测定荧光强度信号,带入实施例1检测的标准曲线得到样品中的AFB1浓度,每份样品重复测定3次取平均值,计算RSD及回收率如下表所示:
经验证所制备的荧光适配体传感器对AFB1的检测具有准确度高,灵敏度好,重现性与稳定性好的特点。同时,对实际样品(如花生)的检测结果表明所制备的传感器具有非常好的实际应用价值。
对于本领域技术人员而言,显然本发明不限于上述示范性实施例的细节,而且在不背离本发明的精神或基本特征的情况下,能够以其他的具体形式实现本发明。因此,无论从哪一点来看,均应将实施例看作是示范性的,而且是非限制性的,本发明的范围由所附权利要求而不是上述说明限定,因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。不应将权利要求中的任何附图标记视为限制所涉及的权利要求。
此外,应当理解,虽然本说明书按照实施方式加以描述,但并非每个实施方式仅包含一个独立的技术方案,说明书的这种叙述方式仅仅是为清楚起见,本领域技术人员应当将说明书作为一个整体,各实施例中的技术方案也可以经适当组合,形成本领域技术人员可以理解的其他实施方式。
Claims (9)
1.一种DNA纳米花原位合成金纳米团簇的制备方法,其特征在于,包括以下步骤:
(1)DNA纳米花的制备:模板链(Padlock)和引物链(Primer)共孵育,在T4 DNA连接酶作用下形成环状模板;将环状模板加入Phi29 DNA聚合酶体系溶液,通过聚合酶驱动滚动循环放大生成具有双功能的DNA纳米花;
(2)原位合成金纳米团簇:将 DNA纳米花加入到柠檬酸钠缓冲液中,并添加四氯金酸溶液,置于磁力搅拌器中搅拌孵育得到荧光信号标签DNA纳米花@AuNCs。
2.根据权利要求1所述的一种DNA纳米花原位合成金纳米团簇的制备方法,其特征在于,所述的双功能DNA纳米花为具有原位孵育金属纳米团簇和核酸识别功能的DNA纳米花。
3. 根据权利要求1所述的一种DNA纳米花原位合成金纳米团簇的制备方法,其特征在于,所述的Padlock的序列为SEQ ID NO.1:5’-P- ATG ATA TGA TCG TTG TCA CTG CCT GCTTTT TTT TTT TAT GAT GG-3’;
SEQ ID NO.2:5’-P- ATG ATA TGA TCG TTG TCA CTG CCT GCT TTT TTT TTT TTT TTTTTT TTA TGA TGG-3’;
SEQ ID NO.3:5’-P- ATG ATA TGA TCG TTG TCA CTG CCT GCT TTT TTT TTT TTT TTTTTT TTT TTT TAT GAT GG-3’;
SEQ ID NO.4:5’-P- ATG ATA TGA TCG TTT TTT TTT TAT GAT CGT TGT CAC TGC CTGCTT TTT TTT TTT ATG ATG G -3’中的一种。
4. 根据权利要求1所述的一种DNA纳米花原位合成金纳米团簇的制备方法,其特征在于,所述的Primer的序列为SEQ ID NO.5所示:具体为5’-CGA TCA TAT CAT CCA TCA TAAAAA-3’。
5. 根据权利要求1所述的一种DNA纳米花原位合成金纳米团簇的制备方法,其特征在于,所述的Phi29 DNA聚合酶体系溶液包括Phi29 DNA聚合酶,Phi29 缓冲液,dNTPs。
6.根据权利要求1所述的一种DNA纳米花原位合成金纳米团簇的制备方法,其特征在于,所述的四氯金酸溶液体系包括四氯金酸(HAuCl4),柠檬酸钠缓冲溶液。
7.一种锰金属有机框架荧光适配体传感器的制备方法,其特征在于,包括以下步骤:
(1)锰金属有机框架(Mn-MOF)负载DNA的制备:将醋酸锰和H2TCPP置于N,N-二甲基甲酰胺溶液中混合均匀,室温孵育后得到Mn-MOF;
(2)使用EDC/NHS溶液活化Mn-MOF表面羧基,与氨基修饰的发夹2(H2)链共孵育,连接后得到Mn-MOF-H2;
(3)将含有目标物的样品制成不同浓度的待测液,加入适配体发夹1(H1)链,Mn-MOF-H2和发夹3(H3)链,通过催化发夹自组装反应,获得大量的Mn-MOF-H2/H3;
(4)将Mn-MOF-H2/H3与权利要求1步骤(2)中制得的DNA纳米花@AuNCs混合孵育后,Mn-MOF上修饰的H3链与DNA纳米花互补结合,随后通过H3链3’端的6G序列淬灭AuNCs的荧光,通过荧光仪测量荧光强度变化,建立荧光信号响应值与目标物浓度间的关系曲线,检测实际样品的信号值,代入关系曲线得到目标物浓度。
8.根据权利要求7所述的一种锰金属有机框架荧光适配体传感器的制备方法,其特征在于,所述的氨基修饰的H2链为3’端氨基化。
9. 根据权利要求7所述的一种锰金属有机框架荧光适配体传感器的制备方法,其特征在于,当所述的目标物为黄曲霉毒素B1时,H1链的序列如SEQ ID NO.6所示:具体为5’-GTTGGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CCA CAC TAA CAT ATTCAA GGG CAC GAG ACA CAG AG-3’;H2的序列如SEQ ID NO.7所示:具体为5’-CTC TGT GTCTCG TGC CCT TGA ATA TGT TAG ATG ATC GTT GTC ACT GCC TGC CTA ACA TAT TCA AGCGAA GCT C-NH2-3’;H3的序列如SEQ ID NO.8所示:具体为5’-TTG AAT ATG TTA GGC AGGCAG TGA CAA CGA TCA TCT AA CAT ATT CAA GGG CAC GAG ACA CAG AGA TGA TCG TTGTCA CTG CCT GCG GGG GG-3’。
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