CN115386105A - 多重酶活性纳米酶荧光水凝胶的制备方法及应用 - Google Patents

多重酶活性纳米酶荧光水凝胶的制备方法及应用 Download PDF

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CN115386105A
CN115386105A CN202211028903.1A CN202211028903A CN115386105A CN 115386105 A CN115386105 A CN 115386105A CN 202211028903 A CN202211028903 A CN 202211028903A CN 115386105 A CN115386105 A CN 115386105A
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杨亚玲
李秋兰
刘佳鑫
杨德志
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Kunming University of Science and Technology
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Abstract

本发明公开了一种多重酶活性纳米酶荧光水凝胶的制备方法,该方法由银纳米Cu,I@AgNPs与Mo,Fe‑CDs组成纳米酶Mo,Fe/Cu,I@Ag,并将葡萄糖氧化酶与该纳米酶负载到水凝胶上;利用水凝胶表现出的GOx、拟过氧化物酶、拟过氧化氢酶和拟超氧化物歧化酶活性与pH响应葡萄糖引发的级联反应用于糖尿病创面愈合;利用含Mo,Fe/Cu,I@Ag@GOx纳米酶通过Ca2+交联海藻酸钠和壳聚糖构建的水凝胶具有可注射性、力学和黏附性以及荧光性质和有益的生物相容性;重要的是,纳米酶凝胶可溶于水或乙醇,在用作敷料时不会对伤口造成二次损伤,本发明水凝胶具有高效促血管生成、细菌感染创面愈合和原位监测等特点。

Description

多重酶活性纳米酶荧光水凝胶的制备方法及应用
技术领域
本发明涉及纳米材料抗菌技术领域,具体为一种多重酶活性纳米酶荧光水凝胶的制备方法及应用。
背景技术
糖尿病创面由于具有持续的葡萄糖积累、持续的细菌感染、受损的血管生成导致创面缺氧和氧化应激等特点,导致创面的慢性愈合。有研究报道基于葡萄糖氧化酶(GOx)和过氧化物酶(POD)的葡萄糖激活级联反应,通过解决高血糖和细菌感染促进伤口修复。GOx与葡萄糖作用产生过氧化氢(H2O2)和葡萄糖醛酸,使伤口部位的pH降低到3~4,提供了POD反应的条件,H2O2通过模拟POD的活性进一步催化生成羟基自由基(·OH),导致葡萄糖消耗和细菌死亡。这样就规避了直接使用有毒的H2O2,以克服pH对拟POD活性的限制。拟超氧化物歧化酶(SOD)纳米酶能将超氧自由基(O2 ·-)转化为H2O2和O2,清除O2 ·-。拟过氧化氢酶(CAT)纳米酶能将H2O2转化为O2,减轻氧化应激和缺氧,使皮肤再生。具有多重模拟酶活性的纳米酶在糖尿病伤口愈合中有非常好的促伤口愈合作用。碳点(CDs)是一种10nm以下超小尺寸的零维纳米材料,由于其优异的光学性能、水溶特性,特别是良好的生物相容性等特点,在抗菌治疗方面具有巨大的潜力。最近的研究表明,金属掺杂碳点会导致POD类催化过程中活性位点的利用率提高,这种杂原子掺杂不仅可以改变CDs的内部电子环境,可以提供活性位点,赋予CDs新的功能。CDs大都具有拟POD活性,很少有研究报道CDs纳米材料具有三重模拟酶活性(包括拟POD、CAT和SOD)。
发明内容
本发明提供了一种多重酶活性纳米酶荧光水凝胶的制备方法,该方法利用了纳米酶的多重纳米酶活性,结合GOx,负载于水凝胶中,利用级联反应,用于糖尿病伤口愈合。
本发明制备了一种由Cu-I掺杂碳点(Cu,I-CDs)还原硝酸银为银纳米(Cu,I@Ag)与Mo、Fe掺杂碳点(Mo,Fe-CDs)组成的具有多酶类活性纳米酶(Mo,Fe/Cu,I@Ag),并将葡萄糖氧化酶(GOx)与该纳米酶负载到水凝胶上,利用水凝胶表现出的GOx、拟过氧化物酶(POD)、拟过氧化氢酶(CAT)和拟超氧化物歧化酶(SOD)活性与pH响应葡萄糖引发的级联反应用于糖尿病创面愈合。第一个级联反应,由GOx引发,纳米酶水凝胶催化葡萄糖和O2转化为葡萄糖酸和H2O2,在POD作用下生成羟基自由基(·OH)来根除细菌;第二个级联反应是,随着创面pH值的变化,碱性微环境发生变化,纳米酶水凝胶模拟SOD将O2 ·-转化为O2和H2O2,再通过拟CAT机制将内源性和外源性H2O2分解成O2,减轻氧化应激,缓解缺氧,促进糖尿病创面愈合;利用Mo,Fe/Cu,I@Ag@GOx纳米酶,通过Ca2+交联海藻酸钠(SA)和壳聚糖(CS)构建的水凝胶具有可注射性、力学和黏附性以及荧光性质和有益的生物相容性;重要的是,纳米酶水凝胶可溶于水或乙醇,在用作敷料时不会对伤口造成二次损伤;多功能荧光水凝胶具有高效促血管生成、促细菌感染创面愈合和原位监测等特点。
本发明多重酶活性纳米酶荧光水凝胶的制备如下:
(1)钼铁掺杂碳点(Mo,Fe-CDs)的合成:称取1.1-1.5g Na2MoO4、1.3-1.5gFeCl3.6H2O、0.03-0.05g多巴胺、2.1-2.5g柠檬酸及0.05-0.1g乙二胺溶于30-40mL超纯水中,超声处理15-20分钟,将溶液转移至聚四氟乙烯内衬不锈钢反应器中,置于马弗炉中,在200℃反应10-12小时,反应完成后自然冷却至室温,得棕色溶液;将棕色溶液用0.22μm滤膜除去大颗粒杂质,再经高速离心,上清液真空干燥,得到Mo,Fe-CDs;
(2)Cu,I掺杂碳点(Cu,I-CDs)的合成:称取0.1-0.3g CuCl2、1.3-1.5mmol/L3-碘-L-酪氨酸及0.1-0.5g乙二胺溶于40-50mL超纯水中,超声处理15-20分钟,将溶液转移至聚四氟乙烯内衬不锈钢反应器中,置于马弗炉中,在180℃反应8-10小时,反应完成后自然冷却至室温,得棕色溶液;将棕色溶液用0.22μm滤膜除去大颗粒杂质,再经高速离心,上清液真空干燥,得到Cu,I-CDs;
(3)银纳米Cu,I@Ag合成:将10-15mg的Cu,I-CDs溶于30mL超纯水中,加入AgNO3,AgNO3在混合液中的浓度为20-25mg/mL,避光搅拌30-60分钟,高速离心,胶体用去离子水洗涤3次,移出多余的Cu,I-CDs和AgNO3,冷冻干燥,得到银纳米Cu,I@Ag;
(4)Mo,Fe/Cu,I@Ag合成:将Mo,Fe-CDs 与Cu,I@Ag按重量比1:1-3的比例加入到超纯水中,混合溶液室温搅拌24小时,高速离心,上清液60℃真空干燥12-24小时制得Mo,Fe/Cu,I@Ag;
(5)Mo,Fe/Cu,I@Ag@GOx水凝胶的合成:将0.5-1.0g海藻酸钠(SA)加入到7-10mL甘油和5-10mL 浓度1-5mg/mLMo,Fe/Cu,I@Ag的混合液中,搅拌混合1-2小时后,加入质量浓度0.8-1.2%的壳聚糖(CS)溶液10-15mL、葡萄糖氧化酶(GOx)0.125-0.5mg和质量浓度1-3%的CaCl2溶液200-500μL,溶胶-凝胶转变24小时,得到Mo,Fe/Cu,I@Ag@GOx水凝胶。
步骤(1)-(4)中高速离心是在10000r/min下处理15-20分钟。
本发明另一目的是将上述方法制得的多重酶活性纳米酶荧光水凝胶应用在制备糖尿病伤口愈合试剂中。
本发明的优点在于:
1、本发明利用纳米酶具有三重模拟酶,包括POD、CAT和SOD活性的特性,将其与天然GOx负载于水凝胶中,在用于糖尿病伤口治疗时,发生级联反应,第一个级联反应,由GOx引发,纳米酶凝胶催化葡萄糖和O2转化为葡萄糖酸和H2O2,POD催化H2O2羟基自由基(·OH)来根除细菌;第二个级联反应是,随着创面pH值的变化,碱性微环境发生变化,纳米酶凝胶模拟SOD将O2 ·-转化为O2和H2O2,通过拟CAT机制将内源性和外源性H2O2分解成O2,减轻氧化应激,缓解缺氧,促进糖尿病创面愈合;
2、利用含Mo,Fe/Cu,I@Ag@GOx纳米酶通过Ca2+交联海藻酸钠(SA)和壳聚糖(CS)构建的水凝胶具有可注射性、力学和黏附性以及荧光性质和有益的生物相容性;
3、纳米酶水凝胶可溶于水或乙醇,在用作敷料时不会对伤口造成二次损伤,多功能荧光水凝胶具有高效促血管生成、细菌感染创面愈合和原位监测等特点;
本发明水凝胶制备工艺简单,适用于工业化生产和市场推广应用。
附图说明
图1为不同水凝胶氧化TMB(POD活性)的紫外可见吸收光谱图,图中(1)为单独TMB,不添加水凝胶的空白对照,(2)Mo,Fe-CDs@GOx水凝胶,(3)Cu,I@Ag@GOx水凝胶,(4)Mo,Fe/Cu,I@Ag@GOx水凝胶;
图2为不同水凝胶过氧化氢酶(CAT)活性结果示意图,图中(1)为不添加水凝胶的空白对照,(2)Mo,Fe-CDs@GOx水凝胶,(3)Cu,I@Ag@GOx水凝胶,(4)Mo,Fe/Cu,I@Ag@GOx水凝胶;
图3为不同水凝胶拟超氧化物歧化酶(SOD)活性结果示意图,图中(1)为不添加水凝胶的空白对照,(2)Mo,Fe-CDs@GOx水凝胶,(3)Cu,I@Ag@GOx水凝胶,(4)Mo,Fe/Cu,I@Ag@GOx水凝胶;
图4为水凝胶的荧光性能图,上图为自然光下的状态,下图为紫外光下的状态;Mo,Fe-CDs gel为Mo,Fe-CDs@GOx水凝胶,Cu,I@Ag gel为Cu,I@Ag@GOx水凝胶,Mo,Fe/Cu,I@Aggel为Mo,Fe/Cu,I@Ag@GOx水凝胶;
图5为水凝胶的注射性能图;
图6为水凝胶的粘附性能图;
图7为水凝胶的自愈性能图;
图8为水凝胶溶解性图;
图9水凝胶的含水量结果,图中水凝胶基底为不添加Mo,Fe/Cu,I@Ag@GOx纳米酶制得的凝胶,Mo,Fe-CDs水凝胶为Mo,Fe-CDs@GOx水凝胶,Cu,I@Ag水凝胶为Cu,I@Ag@GOx水凝胶;
图10为本发明水凝胶细胞毒性实验结果;
图11为不同水凝胶抗菌实验结果,图中Mo,Fe-CDs gel为Mo,Fe-CDs@GOx水凝胶,Cu,I@Ag gel为Cu,I@Ag@GOx水凝胶;
图12为水凝胶对糖尿病小鼠伤口愈合照片;
图13为水凝胶对糖尿病小鼠伤口愈合率统计结果。
具体实施方式
下面将结合具体的实施例对本发明的技术方案作进一步详细地描述说明,但本发明的保护范围并不仅限于此。
实施例1:多重酶活性纳米酶的制备及性能
(1)称取1.2g Na2MoO4、1.3gFeCl3.6H2O、0.03g多巴胺、2.2g柠檬酸及0.06g乙二胺溶于35mL超纯水中,超声处理15分钟,将溶液转移至聚四氟乙烯内衬不锈钢反应器中,置于马弗炉中,200℃反应10小时,反应完成后自然冷却至室温,得棕色溶液;将棕色溶液用0.22μm滤膜除去大颗粒杂质,再在10000r/min下离心15分钟,上清液真空干燥,得到钼铁掺杂碳点Mo,Fe-CDs;
(2)称取0.2g CuCl2、1.3mmol/L 3-碘-L-酪氨酸及0.2g乙二胺溶于40mL超纯水中,超声处理20分钟,将溶液转移至聚四氟乙烯内衬不锈钢反应器中,置于马弗炉中,180℃反应10小时,反应完成后自然冷却至室温,得棕色溶液;将棕色溶液用0.22μm滤膜除去大颗粒杂质,再在10000r/min下离心15分钟,上清液真空干燥,得到Cu,I掺杂碳点Cu,I-CDs;
(3)银纳米Cu,I@Ag合成:将10mg的Cu,I-CDs溶于30mL超纯水中,加入AgNO3,AgNO3在混合液中的浓度为20mg/mL,避光搅拌40分钟,在10000r/min下离心15分钟,胶体用去离子水洗涤3次,移出多余的Cu,I-CDs和AgNO3,冷冻干燥,得到银纳米Cu,I@Ag;
(4)按1:2de 重量比,将Mo,Fe-CDs与Cu,I@Ag加入到超纯水中,混合溶液室温搅拌24小时,在10000r/min下离心15分钟,上清液60℃真空干燥15小时制得Mo,Fe/Cu,I@Ag;
(5)将0.6g海藻酸钠(SA)加入到8mL甘油和6mL浓度2mg/mLMo,Fe/Cu,I@Ag的混合液中,搅拌混合1小时后,加入10mL壳聚糖(CS,1%)、葡萄糖氧化酶(GOx,0.2mg)和250μLCaCl2溶液(2%),溶胶-凝胶转变24小时,得到Mo,Fe/Cu,I@Ag@GOx水凝胶;
(6)采用TMB显色反应测定水凝胶的拟过氧化酶(POD)活性
将100µg/mL 水凝胶100μL、100mmol/L的TMB 50µL、10mmol/L H2O250 μL加入到pH4.5醋酸盐缓冲溶液2mL中,充分混匀,室温孵育10分钟后,离心分离,取上层清液用紫外-可见分光光度计在655nm处测量吸光度,每个样品测量3次,取平均值,结果如图1;从图1中可以看出,在酸性条件下,本实施例制得的水凝胶表现出相当高的拟POD活性;图1中Mo,Fe-CDs水凝胶、Cu,I-CDs水凝胶是在步骤(5)制备过程中用Mo,Fe-CDs、Cu,I-CDs替换Mo,Fe/Cu-I@Ag制得的水凝胶;
(7)采用 TiCl4显色反应测定水凝胶的拟过氧化氢酶(CAT)活性
将100µg/mL水凝胶100μL与1mL、50mmol/L的 H2O2反应10分钟,加入200μL10%TiCl4,充分混匀,室温孵育10分钟后,离心分离,取上层清液用紫外-可见分光光度计在415nm处测量吸光度,每个样品测量3次,取平均值;
结果如图2;从图2中可以看出,本实施例制得的水凝胶表现出好的拟过氧化氢酶活性。
(8)采用氯化硝基蓝四氮唑(NBT)显色反应测定水凝胶的拟超氧歧化物酶(SOD)活性
将100µg/mL水凝胶100μL加入到5mg/mL NBT100μL 和pH=3磷酸盐缓冲液的混合物中,反应15min后,在580nm处测定吸光度,每个样品测量3次,取平均值,结果如图3;从图3中可以看出,本实施例制得的水凝胶表现出相当高的拟SOD活性。
(9)水凝胶的性能测试
Figure 61045DEST_PATH_IMAGE001
荧光性能:凝胶的荧光性质如图4所示,本实施例制得的水凝胶在365nm紫外光下表现出明显的荧光特性,荧光凝胶有望用于原位监测抗菌和伤口愈合过程,凝胶优异的荧光性能归因于碳点。
Figure 959731DEST_PATH_IMAGE002
Mo,Fe/Cu,I@Ag@GOx水凝胶的注射、粘附、自愈性能及溶解测试:如图5所示的凝胶的可注射性,该凝胶可以很容易地吸入到移液管中,并以特定的形状喷射出来,如“KUST”,表明在注射的可持续释放中能应用。凝胶的粘附性如图6所示,Ca2+交联凝胶紧紧地粘附在手指上,即使手指从0~90°弯曲也没有移动,胶黏性归因于凝胶内部的键能。图7显示了凝胶的自愈能力,当凝胶被分成两部分并接触在一起时,凝胶在几秒钟内就恢复为一个整体,可以拉伸而不破裂,这归因于Ca2+与CS及SA共价键的形成,损伤后可修复数秒,图8显示了凝胶在浓度75%的乙醇中有好的溶解性。
Figure 919465DEST_PATH_IMAGE003
不同水凝胶的含水量测试
参照文献(Shengbo Li等,Calcium ion cross-linked sodium alginatehydrogels containingdeferoxamine and copper nanoparticles for diabetic woundhealing.International Journal of Biological Macromolecules 202 (2022) 657–670)中方法进行含水量测定,水凝胶的湿重(W湿)和在真空环境中冻干12小时后的干重(W),根据以下公式计算水凝胶的含水量:含水量(%)=(W湿− W)/ W湿× 100%;结果如图9所示,Mo,Fe/Cu,I@Ag@GOx水凝胶含有95%的水。
Figure 663430DEST_PATH_IMAGE004
细胞毒性测试:采用CCK-8细胞活力试剂盒检测水凝胶的细胞毒性,具体实验,将人脐静脉内皮细胞(HUVECs, 北纳创联生物科技有限公司)接种于96孔板中培养24小时后,分别用浓度为(160μM,以Ag+含量计算)Mo,Fe/Cu,I@Ag@GOx处理12、24、36小时后,分次用PBS漂洗细胞,然后每孔加入CCK-8溶液至10%浓度,37℃孵育,在450nm处测定吸光度;CCK-8分析(图10)显示Mo,Fe/Cu,I@Ag@GOx水凝胶对细胞均无明显毒性。
Figure 775743DEST_PATH_IMAGE005
水凝胶抗菌实验
以下菌种分别从北纳创联生物科技有限公司、云南大学微生物研究所及昆明理工大学生命科学与技术学院获得;
实验方法:以金黄色葡萄球菌(S.aureus,ATCC25923)和铜绿假单胞菌(P.aeruginosa,ATCC27853)为实验菌株。采用平板计数法,通过计数CFU数来判定水凝胶的抗菌性能。首先,将上述菌种在固体Luria-Bertani(LB)培养基和固体营养肉汤培养基孵育24小时,用接种环挑取少量形成的菌落,接种到对应液体培养基(5mL)中,然后,在37℃、180rpm恒温摇床下震荡孵育12小时后,即获得细菌悬浮液(1×108 CFU/mL),用无菌磷酸盐缓冲液(PBS)稀释到1×105 CFU/mL。
实验材料分为四组:空白对照组、Mo,Fe-CDs@GOx水凝胶组、Cu,I-Ag@GOx水凝胶组和Mo,Fe/Cu,I@Ag@GOx水凝胶组,将培养的细菌加入磷酸盐缓冲液中作为空白对照组,其他组将培养的细菌与不同水凝胶混合,水凝胶浓度为100μg/mL,然后在37℃下将孵育60分钟,菌悬液经过稀释后(100μL)均匀涂布在LB固体培养基和营养肉汤固体培养基上,在37℃下培养24小时,计算菌落数,判断抗菌性能。
结果如图11所示,空白对照组几乎没有抗菌性能,Mo,Fe-CDs@GOx水凝胶组对铜绿假单胞菌和金黄色葡萄球菌杀菌率分别为27.3%、37.1%、Cu,I-Ag@GOx水凝胶组对铜绿假单胞菌和金黄色葡萄球的抗菌率分别为57.4%和60.1 %,Mo,Fe/Cu,I@Ag@GOx水凝胶对铜绿假单胞菌和金黄色葡萄球菌有近100%的杀菌率。
(9)小鼠伤口愈合试验
糖尿病小鼠及背部创面模型构建:所有动物实验均符合《动物护理指导原则》。选取6~8周龄、体重18~20g的雄性ICR小鼠作为实验动物。糖尿病伤口准备步骤如下:小鼠喂高脂饲料2周,腹腔注射链脲佐菌素(50mg/kg),链脲佐菌素溶于枸橼酸盐缓冲液(pH4.5)中,每天1次,连续5次注射,1周后通过尾静脉注射法测定小鼠血糖水平。连续两周血糖水平高于16.7mmol/L的小鼠被鉴定为Ⅰ型糖尿病小鼠。接下来,用医用剪刀在乙醚麻醉的小鼠背部切开直径1cm的圆形手术伤口。然后,将100μL金黄色葡萄球菌或铜绿假单胞菌悬液(1×108 CFU/mL)均匀涂于创面,用纱布和医用胶带包扎。小鼠感染菌24小时后,随机分为4组(每组5只小鼠):对照组、Mo,Fe-CDs@GOx水凝胶组、Cu,I@Ag@GOx水凝胶组和Mo,Fe/Cu,I@Ag@GOx水凝胶组,对照组在小鼠伤口处涂抹无菌PBS,其余凝胶组中水凝胶浓度为100μg/mL,采用注射器将上述各组水凝胶(各300μL)注到小鼠伤口处并涂抹均匀,每隔24小时更换一次小鼠创面上的水凝胶,在第0、1、3、5、7天测量小鼠创面情况,通过愈合率(%) = (A0 - At) /(A0×100)计算创面愈合率,其中A0为初始创面面积,At为各时间点的残余创面面积。
实验结果见图12、13,对照组于第1天出现脓液,持续至第7天,提示伤口感染,第7天,对照组的伤口变得更大,而Mo,Fe/Cu,I@Ag@GOx水凝胶组创面面积明显减少,创面愈合率90.15%,在其他治疗组创面面积中最高,创面基本愈合,
以上结果表明,本发明制备的纳米酶水凝胶有多重酶活性,通过级联反应起到抗菌、提供氧及清除自由功能,使具有糖尿小鼠伤口具有好的愈合效果,同时在凝胶好的粘附性能,生物相容性、抗菌性能及伤口愈合性能,其溶解性能在用作敷料时不会对伤口造成二次损伤,多功能荧光水凝胶具有高效促血管生成、细菌感染创面愈合和原位监测等特点。

Claims (3)

1.一种多重酶活性纳米酶荧光水凝胶的制备方法,其特征在于,步骤如下:
(1)钼铁掺杂碳点Mo,Fe-CDs的合成
称取1.1-1.5g Na2MoO4、1.3-1.5gFeCl3.6H2O、0.03-0.05g多巴胺、2.1-2.5g柠檬酸及0.05-0.1g乙二胺溶于30-40mL超纯水中,超声处理15-20分钟后,在马弗炉中200℃下反应10-12小时,反应完成后自然冷却至室温,得棕色溶液;将棕色溶液用0.22μm滤膜除去大颗粒杂质,再经高速离心,上清液真空干燥,即得Mo,Fe-CDs;
(2)Cu,I掺杂碳点Cu,I-CDs的合成
称取0.1-0.3g CuCl2、1.3-1.5mmol/L3-碘-L-酪氨酸及0.1-0.5g乙二胺溶于40-50mL超纯水中,超声处理15-20分钟后置于马弗炉中,180℃反应8-10小时,反应完成后自然冷却至室温,得棕色溶液;将棕色溶液用0.22μm滤膜除去大颗粒杂质,再经高速离心,上清液真空干燥,得到Cu,I-CDs;
(3)银纳米Cu,I@Ag合成
将10-15mg的Cu,I-CDs置于30mL超纯水中,加入AgNO3,AgNO3在混合液中的浓度为20-25mg/mL,避光搅拌30-60分钟,高速离心,胶体用去离子水洗涤3次,移出多余的Cu,I-CDs和AgNO3,冷冻干燥,得到银纳米Cu-I@Ag;
(4)Mo,Fe/Cu-I@Ag合成
按重量比1:1-3的比例,将Mo,Fe-CDs与Cu,I@Ag加入到超纯水中,混合溶液室温搅拌24小时,高速离心,取上清液60℃下真空干燥12-24小时制得Mo,Fe/Cu,I@Ag;
(5)Mo,Fe/Cu,I@Ag@GOx水凝胶的合成
将0.5-1.0g海藻酸钠加入到7-10mL甘油和5-10mL浓度1-5mg/mLMo,Fe/Cu,I@Ag的混合液中,搅拌混合1-2小时后,加入质量浓度0.8-1.2%的壳聚糖溶液10-15mL、葡萄糖氧化酶0.125-0.5mg和质量浓度1-3%的CaCl2溶液200-500μL,溶胶-凝胶转变24小时,得到Mo,Fe/Cu-I@Ag@GOx水凝胶。
2.根据权利要求1所述的多重酶活性纳米酶荧光水凝胶的制备方法,其特征在于:步骤(1)-(4)中高速离心是在10000r/min下处理15-20分钟。
3.权利要求1-2中任一项所述的多重酶活性纳米酶荧光水凝胶的制备方法制得的纳米酶荧光水凝胶在制备糖尿病伤口愈合试剂中的应用。
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