CN106483176B - 一种用于fgfr3-1138g>a基因多态性检测的电化学传感器制备方法 - Google Patents
一种用于fgfr3-1138g>a基因多态性检测的电化学传感器制备方法 Download PDFInfo
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Abstract
本发明涉及软骨发育不全产前诊断基因——成纤维细胞生长因子受体3(FGFR3)基因多态性检测的电化学传感器的制备方法及应用,属于电化学检测技术领域。其特征在于:首先合成得到Hemin‑MOFs复合材料,然后将铂纳米粒子还原在Hemin‑MOFs复合材料上,再使单链DNA信号探针与该复合材料混合,制得生物信号探针;然后通过还原性氧化石墨烯四乙烯五胺,纳米金,亲和素,层层自组装用于生物素化的DNA捕获探针的固定,从而制备了FGFR3‑1138G>A基因多态性检测的电化学传感器,该传感器成功的用于FGFR3基因发生单碱基突变的检测。本发明的优点在于灵敏度高,特异性强,检测迅速,方便。本发明为产前无创诊断软骨发育不全提供了新的检测方法。
Description
技术领域:
本发明涉及一种在临床上产前无创诊断FGFR3-1138G>A基因多态性检测的电化学传感器的制备方法及应用,尤其是基于金属有机框架纳米复合材料作为信号探针制备的夹心型生物传感器,用于检测FGFR3-1138G>A基因多态性,属于电化学检测领域。
背景技术:
软骨发育不全(achondroplasia,ACH)是一种常见的常染色体显性遗传病,多由软骨内骨化缺陷而导致的先天性侏儒病。该病患者虽然智力正常但是在体型外表上有严重的缺陷,因此会对患者心理造成严重的负担,所以对于该病的早期产前基因诊断是很有效的预防软骨发育不全缺陷儿出生的措施。99%的ACH患者是由于FGFR3基因第10外显子1138位G>A突变、1138位G>C突变或1123位G>T突变,其中95%以1138位G>A突变。基于此,基因型可以被简化为通过使用FGFR3-1138G>A多态性作为软骨发育不全产前诊断的主要标志物。
传统对于FGFR3突变基因的检测主要依赖于电泳分离、超声检测、绒毛活检术、羊水细胞培养和胚外体腔穿刺术。然而这些方法具有侵入性,对医生的操作要求高且病人不易接受,因此近些年来基因诊断技术越来越多的应用到产前无创诊断。目前常用的基因诊断主要有PCR-单链构象多态性分析、基因测序和基因芯片技术。然而PCR的方法容易受到生物样品中复杂成分的影响,检测效率低和易出现假阳性而使其应用受到了限制。而基因测序不仅需要专门的仪器设备,训练有素的工作人员而且检测过程繁琐费时,因此不适用于常规临床检测。最为主要的是,患者样品中突变序列相对于高水平野生型序列显得寡不敌众,很难获得较高的特异性。因此制备灵敏度高,特异性强,无创性的检测方法对于低丰度突变基因的检测显得至关重要。近年来,基于纳米材料电化学生物传感器由于简便,快速,成本低,灵敏度高等优势而被广泛应用于生物样品的检测。
在电化学生物分析中,信号放大对于提高DNA传感器的灵敏度是非常重要。近些年来,利用具有催化性质的材料用于信号的放大愈来受到重视。氯高铁血红素(Hemin)由于其模拟过氧化物酶的性质而受到广泛的关注,但是单独的Hemin在中性和碱性环境中不仅分散性差而且催化寿命短,因此需要一个载体来保护Hemin。于是金属有机框架(MOFs)被用来有效的固定和保护Hemin,既可以促进Hemin的分散又可以延长Hemin的催化寿命。为了能进一步增强催化性能,实现更多的信号放大,具有催化性能的铂纳米颗粒(PtNPs)是很好的选择,因为PtNPs也具有很强的催化性能。因此将这三者结合形成的复合材料Hemin-MOFs-PtNPs具有三种材料的优点并且催化性能也是最优的,而且因为有PtNPs的存在,也可以与信号探针结合,通过信号探针与目标序列的特异性结合可以用于构建夹层型生物传感器。
本发明基于Hemin-MOFs-PtNPs纳米复合材料构建生物信号探针,建立了一种FGFR3-1138G>A基因多态性检测的电化学DNA生物传感器的制备方法与应用,为产前无创诊断软骨发育不全提供了新的思路。
发明内容:
本发明的目的是提供一种产前无创诊断软骨发育不全的FGFR3-1138G>A基因多态性检测的电化学DNA生物传感器的制备方法与应用,其特征包括以下步骤:
(1)氯高铁血红素(Hemin)-金属有机框架(MOFs)-铂纳米颗粒(PtNPs)-单链脱氧核糖核酸(ssDNA)检测探针的制备;
(2)建立电化学DNA生物传感器,测定FGFR3-1138G>A基因,绘制标准曲线。
本发明所述Hemin-MOFs-PtNPs-ssDNA复合物的制备过程具体包括以下步骤,其特征在于包括以下步骤:
(1)Hemin-MOFs纳米复合材料的制备:
0.126g 2-氨基对苯二甲酸,0.187g FeCl3·6H2O和0.226g Hemin溶于15mL DMF中,混匀。将上述溶液油浴加热4h,在加热开始后15min时加入200μL的冰醋酸,加热结束后控制溶液降温至室温。所得溶液10000/min离心5分钟,分别用DMF,无水乙醇,超纯水各清洗三次。所得产物真空干燥24h后重新分散在超纯水中形成1mg mL-1体系,备用。
(2)Hemin-MOFs-PtNPs纳米复合材料的制备:
1mL H2PtCl6(1%)加入1mL(1mg mL-1)Hemin-MOFs溶液中,剧烈超声15min。所得溶液置于磁力搅拌器上,在400转/分钟的搅拌下,逐滴加入2mL NaBH4(0.1M)至溶液由棕褐色变为黑色,继续搅拌30min。上述混合溶液10000/min离心5分钟,用超纯水清洗三次。所得产物重新分散在超纯水中,4℃备用。
(3)Hemin-MOFs-PtNPs-ssDNA复合物的制备:
用高于100倍的TCEP室温处理巯基修饰的单链DNA,1h。将处理后的检测探针加入Hemin-MOFs-PtNPs溶液中4℃搅拌过夜后离心,并用PBS(0.1M,pH=7.4)清洗。将合成的Hemin-MOFs-PtNPs-ssDNA重新分散在1mL杂交液中,4℃保存备用。
本发明中所述的建立电化学DNA生物传感器,测定FGFR3-1138G>A基因的DNA片段,绘制标准曲线,其特征在于包括以下步骤:
(1)分别用0.3和0.05μm的Al2O3粉末将电极抛光成镜面,然后分别按超纯水、无水乙醇、超纯水的顺序超声电极各5min,室温干燥备用;
(2)将6μL,2mg mL-1rGO-TEPA滴加在电极表面,室温干燥;
(3)将干燥后的电极浸入1%的氯金酸溶液中,恒压法-0.2V沉积30s。
(4)用超纯水将电极冲洗干净后滴加10μL,1μg mL-1亲和素溶液并置于4℃孵育12h。
(5)用超纯水将孵育后的电极冲洗干净后滴加10μL,1μM生物素标记的DNA捕获探针溶液,4℃孵育12h。
(6)用超纯水将孵育后的电极冲洗干净后滴加6μL,0.25%的BSA溶液室温孵育30min。
(7)将上述BSA封闭后的电极用清洗缓冲液(10mM Na2HPO4,2mM KH2PO4,37mMNaCl,2.7mM KCl,pH 7.4)冲洗干净并在氮气中干燥。
(8)将不同浓度的目标DNA滴加在电极上并置于37℃杂交2h。
(9)在干燥后的电极上滴加10μL检测探针混合液置于37℃孵育2h。
(10)将孵育后的电极用清洗缓冲液冲洗干净后置于氮气中干燥。
(11)将电极置于5mL,0.1M PBS(0.1M Na2HPO4,0.1M KH2PO4,0.1M KCl)中进行表征,每隔100s加入20μL,1.2mM H2O2,测量其计时电流变化电流值。
(12)根据所得电流变化值与FGFR3-1138G>A基因DNA片段浓度呈线性关系,绘制工作曲线。
与现有技术相比,本发明的一种产前无创诊断的FGFR3-1138G>A基因多态性检测的电化学DNA生物传感器的制备方法与应用,其突出的特点是:
(1)将基于Hemin-MOFs-PtNPs的纳米复合材料作为信号探针引入到电化学DNA生物传感器的制备中,不仅有效的提高了材料的催化性能,而且提高了生物分子的固载量,进而提高了电化学DNA生物传感器的灵敏度和生物相容性;
(2)通过使用生物素标记的捕获探针用于识别目标DNA,它不仅确保了捕获探针在亲和素修饰的电极表面固定的良好取向,而且通过增加传感器表面用于分子识别的捕获探针密度进而提高了传感器的灵敏度;
(3)本方法制备的电化学DNA生物传感器可为产前无创诊断软骨发育不全提供新方法,进而拓展产前无创基因诊断在常规临床的应用;
(4)使用完全相同的纳米材料和修饰方法,利用捕获探针,信号探针与目标DNA的特异性识别,只需通过改变探针的核酸序列即可实现多种单基因遗传疾病产前无创诊断的特异性,高灵敏检测,另外,此方法简便,快速,便于实现商品化,从而推进转化医学的发展。
附图说明:
图1为本发明的电化学DNA生物传感器的构建示意图。
图2为本发明的信号探针不同合成步骤的扫描电镜图,EDS图和XPS图。
图3为本发明的电化学DNA生物传感器在检测FGFR3-1138G>A基因多态性时得到的计时电流变化电流与浓度的线性关系,以及传感器的特异性与稳定性。
具体实施方式:
下面结合具体实施例对本发明进行进一步阐述,应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1
步骤1.0.126g 2-氨基对苯二甲酸,0.187g FeCl3·6H2O和0.226g Hemin溶于15mLDMF中,混匀。将上述溶液油浴加热4h,在加热开始后15min时加入200μL的冰醋酸,加热结束后控制溶液降温至室温。所得溶液10000/min离心5分钟,分别用DMF,无水乙醇,超纯水各清洗三次。所得产物真空干燥24h后重新分散在超纯水中形成1mg mL-1体系,得到Hemin-MOFs;
步骤2.1mL H2PtCl6(1%)加入1mL(1mg mL-1)Hemin-MOFs溶液中,剧烈超声15min。所得溶液置于磁力搅拌器上,在400转/分钟的搅拌下,逐滴加入2mL NaBH4(0.1M)至溶液由棕褐色变为黑色,继续搅拌30min。上述混合溶液10000/min离心5分钟,用超纯水清洗三次。所得产物重新分散在超纯水中,4℃备用;
步骤3.用高于100倍的TCEP室温处理巯基修饰的单链DNA,1h。将处理后的检测探针加入Hemin-MOFs-PtNPs溶液中4℃搅拌过夜后离心,并用PBS(0.1M,pH=7.4)清洗。将合成的Hemin-MOFs-PtNPs-ssDNA重新分散在1mL杂交液中,4℃保存备用;
步骤4.分别用0.3和0.05μm的Al2O3粉末将电极抛光成镜面,然后分别按超纯水、无水乙醇、超纯水的顺序超声电极各5min,室温干燥备用;
步骤5.将6μL,2mg mL-1rGO-TEPA滴加在电极表面,室温干燥;
步骤6.将干燥后的电极浸入1%的氯金酸溶液中,恒压法-0.2V沉积30s;
步骤7.用超纯水将电极冲洗干净后滴加10μL,1μg mL-1亲和素溶液并置于4℃孵育12h;
步骤8.用超纯水将孵育后的电极冲洗干净后滴加10μL,1μM生物素标记的DNA捕获探针溶液,4℃孵育12h;
步骤9.用超纯水将孵育后的电极冲洗干净后滴加6μL,0.25%的BSA溶液室温孵育30min;
步骤10.将上述BSA封闭后的电极用清洗缓冲液(10mM Na2HPO4,2mM KH2PO4,37mMNaCl,2.7mM KCl,pH 7.4)冲洗干净并在氮气中干燥;
步骤11.将不同浓度的目标DNA滴加在电极上并置于37℃杂交2h;
步骤12.在干燥后的电极上滴加10μL检测探针混合液置于37℃孵育2h;
步骤13.将孵育后的电极用清洗缓冲液冲洗干净后置于氮气中干燥;
步骤14.将电极置于5mL,0.1M PBS(0.1M Na2HPO4,0.1M KH2PO4,0.1M KCl)中进行表征,每隔100s加入20μL,1.2mM H2O2,测量其计时电流变化电流值;
步骤15.根据所得电流变化值与FGFR3-1138G>A基因DNA片段浓度呈线性关系,绘制工作曲线;测定结果表明FGFR3-1138G>A基因片段浓度在0.1fM-1nM范围内成线性关系,线性相关系数为0.9992,检测限为0.033fM。
步骤16.将本发明上述传感器于4℃保存,间断检测传感器电流响应,储存28天后电流响应仍为初始电流的94.5%,表面传感器具有良好的稳定性;
步骤17.本发明取同一批次制备的DNA生物传感器5支,在相同条件下对100pM的FGFR3-1138G>A基因DNA片段分别进行测定,每一支电极测定3次,结果响应电流的相对标准偏差少于2..76%;同时,取不同批次制备的DNA生物传感器2支,在相同条件下对100pM的FGFR3-1138G>A基因DNA片段分别进行测定,每一支电极测定3次,结果响应电流的相对标准偏差少于2.58%,说明传感器批内及批间差异小,传感器重现性良好。
步骤18.将本发明上述传感器用于检测目标核酸序列,错配碱基,结果错配碱基电流响应相对于目标核酸序列显得微不足道,说明传感器的特异性好,可以很好区分目标序列。
以上所述仅是本发明的优选实施方式,应当指出的是,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提条件下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (3)
1.一种用于FGFR3-1138G>A基因多态性检测的电化学传感器制备方法,其特征在于包括以下步骤:
(1)氯高铁血红素-金属有机框架-铂纳米粒子-单链脱氧核糖核酸检测探针的制备;
(2)建立电化学DNA生物传感器,测定FGFR3-1138G>A基因,绘制标准曲线。
2.一种如权利要求1所述的氯高铁血红素-金属有机框架-铂纳米粒子-单链脱氧核糖核酸复合物的制备过程,其特征在于包括以下步骤:
(1)氯高铁血红素-金属有机框架纳米复合材料的制备:
分别称取0.187g六水三氯化铁,0.226g氯高铁血红素和0.126g 2-氨基对苯二甲酸加入15mL的N,N-二甲基甲酰胺溶液中,混匀,将上述混合溶液置于120℃硅油中加热4h,在开始加热后15min时,向混合溶液中加入200μL的冰醋酸,加热结束后控制溶液降温至室温,所得溶液经10000r/min,离心5min,分别用N,N-二甲基甲酰胺,无水乙醇,超纯水各清洗3次,将所得沉淀置于真空干燥24h后,4℃保存备用;
(2)氯高铁血红素-金属有机框架-铂纳米粒子纳米复合材料的制备:
取1mL,1%氯铂酸溶液加入到制备好的1mL浓度为1mg mL-1的氯高铁血红素-金属有机框架复合材料溶液中,剧烈超声20分钟后,向上述溶液中逐滴缓慢加入2mL浓度为0.1M硼氢化钠同时放在磁力搅拌器上进行搅拌反应30min,所得溶液经10000r/min,离心5min,用超纯水清洗3次;
(3)氯高铁血红素-金属有机框架-铂纳米粒子-单链脱氧核糖核酸复合物的制备:
用高于100倍的三(2-羧乙基)膦盐酸盐室温处理巯基修饰的单链脱氧核糖核酸,1h,将处理后的检测探针加入氯高铁血红素-金属有机框架-铂纳米粒子复合材料溶液中4℃搅拌过夜后离心,并用0.1M,pH=7.4的PBS清洗,将合成的氯高铁血红素-金属有机框架-铂纳米粒子-单链脱氧核糖核酸复合物重新分散在1mL杂交液中,4℃保存备用。
3.一种如权利要求1所述的电化学DNA生物传感器检测方法,其特征在于包括以下步骤:
(1)分别用0.3和0.05μm的氧化铝粉末将电极抛光成镜面,然后分别按超纯水、无水乙醇、超纯水的顺序超声电极各5min,室温干燥备用;
(2)将6μL浓度为2mg mL-1的还原性氧化石墨烯-四乙烯五胺滴加在电极表面,室温干燥;
(3)将干燥后的电极浸入1%的氯金酸溶液中,恒压法-0.2V沉积30s;
(4)用超纯水将电极冲洗干净后滴加10μL浓度为1μg mL-1的亲和素溶液并置于4℃孵育12h;
(5)用超纯水将孵育后的电极冲洗干净后滴加10μL浓度为1μM生物素标记的DNA捕获探针溶液,4℃孵育12h;
(6)用超纯水将孵育后的电极冲洗干净后滴加6μL浓度为0.25%的牛血清白蛋白溶液室温孵育30min;
(7)将上述牛血清白蛋白封闭后的电极用10mM磷酸氢二钠,2mM磷酸二氢钾,37mM氯化钠,2.7mM氯化钾配置的pH=7.4的清洗缓冲液冲洗干净并在氮气中干燥;
(8)将不同浓度的目标DNA滴加在电极上并置于37℃杂交2h;
(9)在干燥后的电极上滴加10μL检测探针混合液置于37℃孵育2h;
(10)将孵育后的电极用清洗缓冲液冲洗干净后置于氮气中干燥;
(11)将电极置于用0.1M磷酸氢二钠,0.1M磷酸二氢钾,0.1M氯化钾配置而成的5mL浓度为0.1M的PBS溶液中进行表征,每隔100s加入20μL浓度为1.2mM过氧化氢溶液,测量其计时电流变化电流值;
(12)根据所得电流变化值与FGFR3-1138G>A基因DNA片段浓度呈线性关系,绘制工作曲线。
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