CN114669312A - 一种整合酶的制备方法 - Google Patents
一种整合酶的制备方法 Download PDFInfo
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- CN114669312A CN114669312A CN202210167673.0A CN202210167673A CN114669312A CN 114669312 A CN114669312 A CN 114669312A CN 202210167673 A CN202210167673 A CN 202210167673A CN 114669312 A CN114669312 A CN 114669312A
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- integrase
- zinc
- glucose
- producing
- porous metal
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Abstract
本发明公开了一种整合酶的制备方法,本发明将包裹介孔硅的锌基沸石咪唑酯骨架材料进行热解,以生成的多孔金属氮碳材料作为基底,利用化学还原法原位负载金纳米粒子,构建了纳米酶;最后,通过静电吸附作用将天然葡萄糖氧化酶与纳米酶偶联得到整合酶。通过该方法不仅可保留纳米酶的催化活性,也能提高天然酶的稳定性与催化活性。另外,制得的整合酶还可以对葡萄糖进行一步检测,操作过程简单迅速、结果灵敏、选择性高、检测限低(0.77μM)、检测范围宽(1~300μM),对灵敏检测血糖表现出良好的应用前景。此外,整合酶还具有饥饿、化学动力学和光热三位一体的肿瘤治疗作用。
Description
技术领域
本发明涉及分析化学和纳米药物的制备方法,特别涉及一种整合酶的制备方法。
背景技术
当前,糖尿病由于患病范围广、并发症危害大,成为继肿瘤、心脑血管病之后第三种严重危害人类健康的慢性疾病。为了预防和治疗糖尿病,精确测定血液及体液中葡萄糖的含量具有重要的现实意义。在生物体内,葡萄糖在氧气存在时能被葡萄糖氧化酶催化氧化,产生葡萄糖酸和过氧化氢。因此,目前最常见的检测葡萄糖的方法是采用辣根过氧化物酶定量检测葡萄糖的代谢产物过氧化氢。然而,这个检测过程涉及到葡萄糖氧化酶和辣根过氧化物酶,天然酶性质不稳定性,在催化体系中易失活,且回收困难,不可循环利用。此外,葡萄糖氧化酶和辣根过氧化物酶需要在不同的pH条件下实现其最优催化效率(Small.2018,14,1803256),因此检测需要两步反应实现,步骤繁琐且耗时。
发明内容
发明目的:本发明提供一种对过氧化氢催化性能优异,同时也能提高天然酶的催化活性与稳定性的整合酶的制备方法。
技术方案:本发明所述的整合酶的制备方法,包括以下步骤:
(1)制备锌基沸石咪唑酯骨架材料并在其表面包裹介孔硅;
(2)将得到的骨架材料置于管式炉中煅烧后,经氢氟酸刻蚀,得到多孔金属氮碳材料;
(3)将氯金酸溶液与多孔金属氮碳材料充分混合,利用化学还原法得到纳米酶;
(4)将纳米酶和葡萄糖氧化酶静电偶联得到整合酶。
本发明研究发现,将锌基沸石咪唑酯骨架材料热解生成的金属氮碳材料作为基底,原位负载金纳米粒子制备具有类过氧化物酶活性的纳米酶。热解之前,在锌基沸石咪唑酯骨架外包裹介孔硅,利用介孔硅的物理屏障保护作用,降低材料的表面能,防止煅烧过程中发生团聚;同时,煅烧过程增加材料的石墨化程度,改变材料的电荷分布。通过溶液浸渍法,将氯金酸原位还原在多孔金属氮碳材料上,形成金纳米粒子,并且在金纳米粒子与多孔金属氮碳材料之间形成了金-氮键,提高金纳米粒子的分散性和稳定性,降低金纳米粒子的粒径。多孔金属氮碳材料与金纳米粒子之间的协同作用、金纳米粒子的小尺寸效应,以及多孔氮碳材料的限域效果与底物富集效应共同增强了纳米酶的类过氧化物酶活性。动力学数据表明,纳米酶对氧化底物3,3',5,5'-四甲基联苯胺的亲和力甚至优于天然辣根过氧化物酶。依据纳米酶与葡萄糖氧化酶之间的级联反应,将二者通过静电吸附作用进行近距离组装得到整合酶。得到的整合酶既保留了纳米酶的高催化性能,又提高了葡萄糖氧化酶的稳定性与催化活性。
优选的,步骤(1)中,所述锌基沸石咪唑酯骨架合成材料的原料包括:金属盐类为:六水合硝酸锌或无水醋酸锌;有机配体为:2-甲基咪唑、2-乙基咪唑或苯并咪唑和咪唑-2-甲醛;溶剂为:甲醇、水或N,N-二甲基甲酰胺;所述金属盐和有机配体的摩尔比为1:1~1:70,反应时间为0.5~48h,搅拌速度为400~1200rpm;离心转速为6000~13000rpm,洗涤溶剂为甲醇和水,洗涤三次及以上,产物真空干燥温度为60~120℃,干燥时间为6~24h。
优选的,步骤(1)中,所述由介孔硅包裹的沸石咪唑酯骨架材料原料包括正硅酸四乙酯或硅酸异丙酯的碱性溶液;表面活性剂为十六烷基三甲基溴化铵、十六烷基三甲基氯化铵或十二烷基硫酸钠;溶剂为甲醇和水的混合液,甲醇与水的体积比为5:95~20:80;将步骤(1)所得的锌基沸石咪唑酯骨架溶于甲醇与水混合液中,用1M的氢氧化钠调节pH为9~12,加入表面活性剂用量为0.1~0.5g,硅源用量为0.5~3mL,锌基沸石咪唑酯骨架用量与表面活性剂用量之比为1:1~15:1;反应时间为1~6h,搅拌速度为500~1200rpm;离心转速为6000~13000rpm,洗涤溶剂为甲醇,洗涤三次及以上,产物真空干燥温度为60~120℃,干燥时间为6~24h。
优选的,步骤(2)中,所述煅烧为在氮气氛下,煅烧温度600~1100℃,煅烧时间为1~5h,升温速率为1~10℃/min。
优选的,步骤(2)中,所述刻蚀液体为氢氟酸与水的混合液,氢氟酸与水的体积比为1:99~10:90;刻蚀时间为10~60分钟;经抽滤,水洗至中性;产物真空干燥温度为60~120℃,干燥时间为6~24h。
优选的,步骤(3)中,所述多孔金属氮碳材料与氯金酸溶液的质量比30:1~1:1,溶剂为水;在体系中加入硼氢化钠、单宁酸、抗坏血酸或对苯二酚还原剂,浓度为0.5~5mg/mL,还原剂与氯金酸的摩尔比为1:1~100:1;反应时间为10~360分钟,搅拌速度为500~1200rpm;经抽滤,水洗三次及以上,产物真空干燥温度为60~120℃,干燥时间为6~24h。
优选的,步骤(4)中,所述葡萄糖氧化酶浓度为0.2~5mg/mL,纳米酶浓度为0.5~5mg/mL,溶剂为三(羟甲基)氨基甲烷-盐酸缓冲溶液、磷酸缓冲溶液或去离子水;摇床震荡温度为25~37℃,震荡时间为24~48h;离心转速为6000~13000rpm,洗涤溶剂为水,洗涤三次及以上。
本发明制备的整合酶可以应用在葡萄糖的检测中。具体为:利用葡萄糖氧化酶和纳米酶之间的级联反应,采用紫外分光光度法检测葡萄糖;所述级联反应体系pH为2.5~6.5,整合酶浓度为20~100μg/mL,显色底物3,3',5,5'-四甲基联苯胺浓度为1~100mM,葡萄糖浓度为1~1000μM,反应温度为25~45℃,反应时间为10-100分钟;人体血清稀释10倍检测其中葡萄糖,其它条件相同。
本发明研究发现,利用整合酶构建的传感器对葡萄糖检测,具有较低的检测限(0.77μM)、较宽的检测范围(1~300μM)、选择性好、灵敏度高、操作过程简便,抗干扰能力强、可循环利用,可以一步实现血液中葡萄糖的检测。
同时本发明制备的整合酶可以用于制备治疗肿瘤药物。具体为:整合酶的加药浓度为20~100μg/mL,孵育时间为2~48小时,在808nm近红外光下进行照射,光照时间为0~400秒。
本发明研究发现,整合酶作为纳米材料,可以通过高渗透长滞留效应在肿瘤中积累。整合酶内的葡萄糖氧化酶可以消耗肿瘤细胞内的葡萄糖,通过断绝肿瘤细胞的营养物质供给实现肿瘤的饥饿疗法。同时,整合酶中的纳米酶能催化葡萄糖的代谢产物过氧化氢原位产生羟基自由基,进一步加剧肿瘤细胞的毒性损伤,促进肿瘤细胞凋亡,对肿瘤细胞产生化学动力学治疗效果。此外,整合酶内的金纳米粒子能产生局域等离子体共振效应。金属纳米粒子的大小、形貌、与周围介质的相互作用均可影响金属表面电荷的分布,从而使表面自由电子具有不同的振动频率。本发明制备的金纳米粒子粒径为5nm左右,与载体形成金-氮键,改变了金属表面电子密度,使表面自由电子振动频率与808nm激光发生共振耦合,引起周围温度升高,产生不可逆的细胞损伤和随后的肿瘤细胞消融,所以特别适合治疗浅表肿瘤。因此,本发明制备的整合酶可以实现对肿瘤的饥饿、化学动力学、光热疗法三位一体的协同治疗作用。
有益效果:与现有技术相比,本发明具有以下显著效果:1、本发明制备方法简单,将包裹介孔硅的锌基沸石咪唑酯骨架材料进行热解,以生成的多孔金属氮碳材料作为基底,利用化学还原法原位负载金纳米粒子,构建了纳米酶。在金纳米粒子与多孔金属氮碳材料之间形成的金-氮键,提高了金纳米粒子的分散性和稳定性,降低了金纳米粒子的粒径,使其表现出优异的类过氧化物酶活性。而且,制备的纳米酶具有很好的生物相容性,对细胞没有毒害作用。通过静电吸附作用将天然葡萄糖氧化酶与纳米酶近距离组装得到的整合酶,不仅保留了纳米酶的催化活性,还提高了天然酶的催化活性与稳定性;2、本发明制备的整合酶可以一步实现血液中葡萄糖的检测,操作过程简便,结果灵敏,可循环利用、灵敏度高、抗干扰能力强;3、本发明制备的整合酶具有饥饿、化学动力学、光热疗法三位一体的协同治疗作用,实现抗肿瘤治疗;与传统的肿瘤治疗方式(如手术,放疗和化学疗法)相比,无需引入抗肿瘤药物,采用红外光刺激,副作用小、操作简单。
附图说明
图1为本发明实例1中制备的锌基类沸石咪唑酯骨架和介孔硅包裹锌基类沸石咪唑酯骨架的X射线衍射图;
图2为本发明实例1中制备纳米酶的X射线衍射图;
图3为pH对纳米酶的紫外强度的影响图;
图4为pH对整合酶的紫外强度的影响图;
图5为对葡萄糖检测的线性范围;
图6为对葡萄糖检测的线性范围;
图7为对葡萄糖检测的线性范围;
图8为对葡萄糖检测的特异性;
图9为整合酶的光热转换效应图;
图10为人肝癌细胞在药物作用下,细胞存活率与浓度关系图。
具体实施方式
下面通过实施例进一步说明本发明的技术方案。
实施例1
(1)沸石咪唑酯骨架材料制备:将9.5197g的六水合硝酸锌和3.0769g的2-甲基咪唑分别溶于500mL和400mL的甲醇中,溶解完全后,混合,室温下,1000rpm搅拌2小时。13000rpm下离心,甲醇洗涤三次,60℃真空干燥过夜。
(2)介孔硅包裹的沸石咪唑酯骨架材料制备:将2.6g沸石咪唑酯骨架材料分散于24mL的甲醇中。加入216mL的蒸馏水(总体积240mL,10vol%甲醇)。加入1M的氢氧化钠进行调节至pH为11。加入0.2016g的十六烷基三甲基溴化铵搅拌30分钟。1000rpm搅拌下加入1.2mL的正硅酸四乙酯,搅拌30分钟。13000rpm下离心,甲醇洗涤三次。60℃真空干燥过夜。
(3)多孔金属氮碳材料制备:将干燥好的介孔硅包裹的沸石咪唑酯骨架材料置于10mL陶瓷坩埚中,放于管式炉内。在N2气氛下,以10℃/min的升温速度升温至目标温度600℃。在目标温度下煅烧1h。将上述煅烧后材料用10vol%氢氟酸刻蚀30分钟。抽滤至滤液为中性。60℃真空干燥过夜。
(4)纳米酶制备:100mg多孔金属氮碳材料溶于20mL的蒸馏水中,超声10分钟。加入50μL浓度为51.8mM氯金酸溶液,600rpm下搅拌1h后,快速加入2.6mL新鲜制备的硼氢化钠溶液(1.5mg/mL)再搅拌1h。抽滤,蒸馏水洗涤三次。60℃真空干燥过夜。
(5)整合酶制备:将200μL,5mg/mL的纳米酶,200μL三(羟甲基)氨基甲烷-盐酸缓冲液与100μL,0.2mg/mL的葡萄糖氧化酶混合,室温下震荡过夜、12000rpm离心、蒸馏水洗涤三次。最后分散于500μL的蒸馏水中,置于4℃的冰箱备用。
实施例1的锌基类沸石咪唑酯骨架和介孔硅包裹锌基类沸石咪唑酯骨架以及纳米酶的X射线衍射图分别为图1、2。由图1、2可知,锌基类沸石咪唑酯骨架在包硅后并没有改变材料的晶型。纳米酶的X射线衍射图中在20-30°范围内有石墨碳峰出现,说明氮碳材料成功制备,并且金的衍射峰位置与标准图谱一致,表明金纳米粒子成功负载在氮碳载体上。
实施例2
(1)沸石咪唑酯骨架材料制备:将9.5197g的六水合硝酸锌和3.0769g的2-甲基咪唑分别溶于500mL和400mL的甲醇中,溶解完全后,混合,室温下,1000rpm搅拌2小时。13000rpm下离心,甲醇洗涤三次,60℃真空干燥过夜。
(2)介孔硅包裹的沸石咪唑酯骨架材料制备:将2.6g沸石咪唑酯骨架材料分散于24mL的甲醇中。加入216mL的蒸馏水(总体积240mL,10vol%甲醇)。加入1M的氢氧化钠进行调节至pH为11。加入0.2016g的十六烷基三甲基溴化铵搅拌30分钟。1000rpm下下加入1.2mL的正硅酸四乙酯,搅拌30分钟。13000rpm下离心,甲醇洗涤三次。60℃真空干燥过夜。
(3)多孔金属氮碳材料制备:将干燥好的介孔硅包裹的沸石咪唑酯骨架材料置于10mL陶瓷坩埚中,放于管式炉内。在N2气氛下,以10℃/min的升温速度升温至目标温度1100℃。在目标温度下煅烧5h。将上述煅烧后材料用10vol%氢氟酸刻蚀30分钟。抽滤至滤液为中性。60℃真空干燥过夜。
(4)纳米酶制备:100mg多孔金属氮碳材料溶于20mL的蒸馏水中,超声10分钟。加入50μL浓度为51.8mM氯金酸溶液,600rpm下搅拌1h后,快速加入2.6mL新鲜制备的硼氢化钠溶液(1.5mg/mL)再搅拌1h。抽滤,蒸馏水洗涤三次。60℃真空干燥过夜。
(5)整合酶制备:将200μL,5mg/mL的纳米酶,200μL三(羟甲基)氨基甲烷-盐酸缓冲液与100μL,5mg/mL的葡萄糖氧化酶混合,室温下震荡过夜、12000rpm离心、蒸馏水洗涤三次。最后分散于500μL的蒸馏水中,置于4℃的冰箱备用。
实施例3
(1)沸石咪唑酯骨架材料制备:将9.5197g的六水合硝酸锌和3.0769g的2-甲基咪唑分别溶于500mL和400mL的甲醇中,溶解完全后,混合,室温下,1000rpm搅拌2小时。13000rpm下离心,甲醇洗涤三次,60℃真空干燥过夜。
(2)介孔硅包裹的沸石咪唑酯骨架材料制备:将2.6g沸石咪唑酯骨架材料分散于24mL的甲醇中。加入216mL的蒸馏水(总体积240mL,10vol%甲醇)。加入1M的氢氧化钠进行调节至pH为11。加入0.2016g的十六烷基三甲基溴化铵搅拌30分钟。1000rpm下下加入1.2mL的正硅酸四乙酯,搅拌30分钟。13000rpm下离心,甲醇洗涤三次。60℃真空干燥过夜。
(3)多孔金属氮碳材料制备:将干燥好的介孔硅包裹的沸石咪唑酯骨架材料置于10mL陶瓷坩埚中,放于管式炉内。在N2气氛下,以10℃/min的升温速度升温至目标温度1100℃。在目标温度下煅烧1h。将上述煅烧后材料用1vol%氢氟酸刻蚀30分钟。抽滤至滤液为中性。60℃真空干燥过夜。
(4)纳米酶制备:100mg多孔金属氮碳材料溶于20mL的蒸馏水中,超声10分钟。加入50μL浓度为51.8mM氯金酸溶液,600rpm下搅拌1h后,快速加入2.6mL新鲜制备的硼氢化钠溶液(1.5mg/mL)再搅拌1h。抽滤,蒸馏水洗涤三次。60℃真空干燥过夜。
(5)整合酶制备:将200μL,0.5mg/mL的纳米酶,200μL三(羟甲基)氨基甲烷-盐酸缓冲液与100μL,5mg/mL的葡萄糖氧化酶混合,室温下震荡过夜、12000rpm离心、蒸馏水洗涤三次。最后分散于500μL的蒸馏水中,置于4℃的冰箱备用。
实施例4
纳米酶与整合酶pH优化,具体步骤如下:加入400μL不同pH(3.6~6.5)醋酸-醋酸钠缓冲溶液,10μL浓度为10mM的3,3',5,5'-四甲基联苯胺,20μL实例1中制备的纳米酶,20μL浓度为10mM的过氧化氢。37℃下反应15分钟,过滤,测量紫外吸光度。如图3所示,紫外吸光度在pH为4时达到最大。
加入400μL不同pH(3.6~6.5)醋酸-醋酸钠缓冲溶液,30μL浓度为10mM的3,3',5,5'-四甲基联苯胺,20μL实例1中制备的整合酶,50μL浓度为10mM的葡萄糖。37℃下反应60分钟,过滤,测量紫外吸光度。如图4所示,紫外吸光度在pH为4时达到最大。
图3和4结果说明纳米酶和整合酶反应最优pH一致,实现了对于过氧化氢和葡萄糖的一步检测。
实施例5
葡萄糖检测具体步骤如下:
加入400μL pH为4的醋酸-醋酸钠缓冲溶液,30μL浓度为10mM的3,3',5,5'-四甲基联苯胺,20μL实例1中制备,浓度为80μg/mL,酶负载量为1mg/mL的整合酶,不同浓度的葡萄糖(0~500μM)50μL。37℃下反应60分钟,过滤,测量紫外吸光度。如图5-7所示,紫外吸光度值与葡萄糖浓度在1~300μM呈线性关系,检测限为0.77μM。
实施例6
对葡萄糖检测的特异性:
加入400μL pH为4的醋酸-醋酸钠缓冲溶液,30μL浓度为10mM的3,3',5,5'-四甲基联苯胺,20μL实例1中制备,浓度为80μg/mL,酶负载量为1mg/mL的整合酶,10mM的葡萄糖50μL。不同物质(麦芽糖、木糖、果糖、半乳糖、K+、Na+)代替葡萄糖,浓度为葡萄糖的5倍。37℃下反应60分钟,过滤,测量紫外吸光度。如图8所示,紫外吸光度在葡萄糖处显示最大,说明对葡萄糖检测具有特异性。
实施例7
血糖检测具体步骤如下:
加入400μL pH为4的醋酸-醋酸钠缓冲溶液,30μL浓度为10mM的3,3',5,5'-四甲基联苯胺,20μL实例1中制备,浓度为80μg/mL,酶负载量为1mg/mL的整合酶,稀释10倍的血清50μL,37℃下反应60分钟,过滤,测量紫外吸光度。
如表1所示,取线性范围内低中高三个浓度进行回收率测定,与标准曲线相比,其回收率达到95.40%~98.30%,相对标准偏差达到3.1%~6.3%,在误差允许范围内。表明整合酶对实际样品检测的可行性。
表1检测人体血清中葡萄糖的回收率
实施例8
光热治疗具体步骤如下:
取1mL不同浓度的整合酶,在808nm近红外激光进行照射。以磷酸缓冲溶液作为空白对照组。记录升温速率。如图9所示,整合酶具有光热转换能力,且升温速率具有浓度依赖性。
实施例9
饥饿和化学动力学治疗具体步骤如下:
在96孔板中加入人肝癌细胞,每个孔的细胞个数约为5×103,加药浓度为20~100μg/mL的整合酶4μL,37℃孵育2~48h,0.5mg/mL的噻唑蓝200μL,150μL二甲基亚砜。采用噻唑蓝比色法在490nm处测定其光吸收度,确定细胞存活率。如图10所示,整合酶在2μg/mL浓度下即显示出对癌细胞的杀伤能力。
Claims (9)
1.一种整合酶的制备方法,其特征在于:包括以下步骤:
(1)制备锌基沸石咪唑酯骨架材料并在其表面包裹介孔硅;
(2)将得到的骨架材料置于管式炉中煅烧后,经氢氟酸刻蚀,得到多孔金属氮碳材料;
(3)将氯金酸溶液与多孔金属氮碳材料充分混合,利用化学还原法得到纳米酶;
(4)将纳米酶和葡萄糖氧化酶静电偶联得到整合酶。
2.根据权利要求1所述的整合酶的制备方法,其特征在于:步骤(1)中,所述锌基沸石咪唑酯骨架合成材料的原料包括:金属盐类、有机配体、溶剂。
3.根据权利要求2所述的整合酶的制备方法,其特征在于:所述金属盐为六水合硝酸锌或无水醋酸锌;有机配体为2-甲基咪唑、2-乙基咪唑或苯并咪唑和咪唑-2-甲醛;溶剂为:甲醇、水或N,N-二甲基甲酰胺。
4.根据权利要求3所述的整合酶的制备方法,其特征在于:金属盐和有机配体的摩尔比为1:1~1:70。
5.根据权利要求1所述的整合酶的制备方法,其特征在于:步骤(1)中,所述介孔硅包裹的锌基沸石咪唑酯骨架材料的原料为正硅酸四乙酯或硅酸异丙酯的碱性溶液、十六烷基三甲基溴化铵或十六烷基三甲基氯化铵或十二烷基硫酸钠、甲醇和水的混合液。
6.根据权利要求1所述的整合酶的制备方法,其特征在于:步骤(2)中,所述煅烧为在氮气氛下,煅烧温度为600~1100℃,煅烧时间为1~5h,升温速率为1~10℃/min。
7.根据权利要求1所述的整合酶的制备方法,其特征在于:步骤(2)中,所述刻蚀的液体为氢氟酸与水的混合液,氢氟酸与水的体积比为1:99~10:90。
8.根据权利要求1所述的整合酶的制备方法,其特征在于:步骤(3)中,所述氯金酸溶液与多孔金属氮碳材料的质量比1:99~30:70。
9.根据权利要求1所述的整合酶的制备方法,其特征在于:步骤(4)中,所述葡萄糖氧化酶浓度为0.2~5mg/mL,纳米酶浓度为0.5~5mg/mL。
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