CN115121251B - 一种磁性FeCo双金属碳基多孔纳米酶的制备方法及应用 - Google Patents
一种磁性FeCo双金属碳基多孔纳米酶的制备方法及应用 Download PDFInfo
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- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J23/00—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00
- B01J23/70—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00 of the iron group metals or copper
- B01J23/74—Iron group metals
- B01J23/75—Cobalt
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Abstract
本发明涉及碳基纳米酶技术领域,具体涉及一种磁性FeCo双金属碳基多孔纳米酶的制备方法及应用。步骤1:利用碎纸屑、浓盐酸、NaOH和尿素水溶液制备废纸桨混合物;步骤2:将铁盐和钴盐溶解于乙醇溶液中,加入废纸桨混合物,经过搅拌、热聚合反应、煅烧和洗涤后制备得到磁性FeCo双金属碳基多孔纳米酶。本发明经过浸渍促使金属铁、钴离子配位改性废纸桨形成类金属有机骨架配合物材料,同时钠离子和氯离子结合为氯化钠晶体模板,制备成一系列高比表面积、大孔容量及多级孔结构的磁性CoFe2O4/多孔碳基纳米酶。所用原料廉价易得,绿色环保,制备策略简单、条件温和,可用于高选择性比色分析或智能手机分析生物流体中H2O2、葡萄糖和谷胱甘肽。
Description
技术领域
本发明涉及碳基纳米酶技术领域,具体涉及一种磁性FeCo双金属碳基多孔纳米酶的制备方法及应用。
背景技术
天然酶因具有高催化活性、底物专一性、催化多样性以及活性可调节等特点,已在疾病诊断与治疗和环境科学等领域受到了广泛关注。但天然酶存在稳定性差、储存条件苛刻、价格昂贵以及大尺寸应用困难等固有缺陷,这些弊端极大地限制了其在生物医学、食品安全以及环境保护等领域的实际应用。
因此,利用生物或化学策略模拟天然酶不但具有重要的科学意义,而且具有广阔的实际应用价值。近年来,研究者发现某些纳米材料自身具有内在的模拟一种或多种生物酶催化活性的能力,它们被科学家称为纳米模拟酶,简称纳米酶。纳米酶的起源可追溯至2007年,我国科学家阎锡蕴院士团队首次发现Fe3O4纳米颗粒自身具有较好的类似过氧化物酶的催化活性,此后纳米酶的研究在全世界科研界迅速崛起。与天然酶或者传统的模拟酶相比,纳米酶具有可操作性强、易于制备、催化活性可调、成本低廉、稳定性高以及功能功能多样化等优点。加之纳米材料自身所具备的优异理化性质(如高比表面、大孔容量以及光学、电学、磁学、热学和力学等性能)进一步赋予了纳米酶多种多样的功能。目前纳米酶在生物、医学、农业、环境治理等多个领域越来越受到人们的广泛关注。
在过去的几年中,纳米酶的研究取得了快速的进展,包括纳米酶的设计合成、纳米酶的类酶催化反应类型、调控类酶催化活性、揭示催化机理和扩展应用等。为了获得功能多样化的纳米酶,科学家们相继探索了金属基纳米酶、碳基纳米酶、金属有机骨架材料纳米酶以及复合材料类纳米酶等纳米模拟酶。相比之下,碳基纳米酶具有高比表面积、大孔容量、可调节的催化活性和形貌等独特的优势,已在传感分析、抗氧化、环境污染物降解、疾病治疗等领域展现了巨大的应用前景。大多数情况下,利用碳基纳米酶固有的类过氧化物模拟酶活性催化双氧水产生羟基自由基从而实现目标分析物的比色分析和特定疾病治疗的目的。
然而,碳纳米酶作为一种无金属催化剂往往存在催化活性低、制备成本高且难以实现循环利用的缺陷,进而导致实际应用效果不是很理想。为了处理这些挑战,科学家们发现碳骨架掺杂单金属或双金属纳米粒子可有效改善碳基纳米酶的催化活性。然而,目前制备碳基纳米酶的碳源多采用昂贵和毒性的化学试剂为原料,大尺寸制备较为困难,特别是大尺寸合成条件下所制备多孔碳材料的高比表面积与大孔容量会急剧下降。因此,这种方法在大规模的酶工业化生产中存在一定局限性。
近年来利用纤维素类废弃物替代传统化学试剂合成碳基纳米材料已是一个趋势,由于其具有廉价易得、毒性低以及含多种生物高分子聚合物的特性。目前,利用生物质衍生碳纳米酶的研究虽有报道,然采用低成本纤维素类废弃物制备高附加值碳基纳米酶仍处于空白。因此,利用纤维素类废弃物为原料,通过低温冷冻处理技术将废纸加工为废纸浆,进一步将其转化为磁性多孔碳基纳米酶有望解决目前化学试剂衍生碳基纳米酶制备成本高、催化活性低以及难以实现大尺寸制备的瓶颈问题。
发明内容
针对上述存在的技术问题,本发明提出了一种磁性FeCo双金属碳基多孔纳米酶制备方法,该方法以纤维素类废纸取代化学试剂可降低碳基纳米酶碳前驱体的成本;尿素/氢氧化钠的引入不但充当废纸的冻溶剂,而且也是合成CoFe2O4纳米粒子的必要试剂,加之铁钴盐释放的氯离子与混合物中的钠离子可形成NaCl纳米微晶模板,达到“一石三鸟”制备磁性多孔碳纳米酶的目标。
为了实现上述目的,本发明所采用的技术方案如下:
一种磁性FeCo双金属碳基多孔纳米酶的制备方法,包括以下步骤:
步骤1:利用碎纸屑、浓盐酸、NaOH和尿素水溶液制备废纸桨混合物;
步骤2:将铁盐和钴盐溶解于乙醇溶液中,加入步骤1制备的废纸桨混合物,经过搅拌、热聚合反应、煅烧和洗涤后制备得到磁性FeCo双金属碳基多孔纳米酶。
优选的,所述步骤1的具体步骤为:
步骤1.1:将剪碎的碎纸屑浸泡于浓盐酸处理;
步骤1.2:将步骤1.1浸泡后的碎纸屑,经洗涤、干燥后进一步浸渍于 NaOH和尿素水溶液中,然后置于-18℃冷冻24小时,解冻后得到废纸桨混合物。
优选的,所述步骤2的具体步骤为:
步骤2.1:将铁盐和钴盐完全溶解于90%乙醇溶液中,加入步骤1制备的废纸桨混合物,常温搅拌1~4小时;
步骤2.2:将步骤2.1的产物转入水热反应釜加热,进行热聚合反应;
步骤2.3:将步骤2.2热聚合反应后的产物在惰性气体保护下,进行煅烧处理,然后依次用蒸馏水、无水乙醇洗涤产物,最后进行干燥处理,得到磁性FeCo双金属碳基多孔纳米酶。
优选的,所述步骤1.1的浓盐酸为38wt%,处理时间为24小时,其中碎纸屑和浓盐酸的配比为5g:2ml。
优选的,所述步骤1.2的NaOH溶液质量分数为7%,尿素溶液质量分数为12%,其中处理后的碎纸屑与NaOH和尿素混合水溶液的比例为 1g:1ml。
优选的,所述步骤2.1中,铁盐和钴盐中Fe(III)与Co(II)的摩尔比为 1.67。
优选的,所述步骤2.1中,所述铁盐为六水合三氯化铁或九水硝酸铁或者无水氯化铁中的一种,所述钴盐为六水合二氯化钴或硝酸钴或醋酸钴中的一种。
优选的,所述步骤2.2中水热反应釜温度为160~180℃,热聚合反应时间为12~24小时。
优选的,所述步骤2.3的煅烧的温度为550℃,时间为3小时。
制备的磁性FeCo双金属碳基多孔纳米酶,应用于选择性比色传感生物流体中H2O2、葡萄糖和谷胱甘肽。
与现有技术相比,本发明的有益效果是:
本发明以废纸为原料,利用浓盐酸预处理并结合尿素/氢氧化钠水溶液中低温冷冻水解技术制得废纸桨,将废纸桨浸泡于一定浓度的水合三氯化铁和氯化钴的乙醇水溶液中,经过浸渍促使金属铁、钴离子配位改性废纸桨形成类金属有机骨架配合物材料,同时将碱性体系中的钠离子和氯离子结合为氯化钠晶体模板,然后通过水热预碳化和惰性气体高温碳化法,制备成高比表面积、大孔容量及多级孔结构的磁性CoFe2O4/多孔碳基纳米酶。
本发明所用原料廉价易得,绿色环保,制备策略简单、条件温和,所得磁性CoFe2O4/多孔碳复合材料质量稳定,作为一种新颖的类过氧化物酶用于高选择性比色分析或智能手机分析生物流体中H2O2、葡萄糖和谷胱甘肽,在进一步成为医学检测领域的类过氧化物模拟酶有重要的价值。
附图说明
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一起用于解释本发明,并不构成对本发明的限制。
在附图中:
图1是实施例制备的磁性FeCo双金属碳基多孔纳米酶的透射电镜图。
图2是实施例制备的磁性FeCo双金属碳基多孔纳米酶与废纸衍生多孔碳的XRD图。
图3是实施例制备的磁性FeCo双金属碳基多孔纳米酶的磁滞曲线图。
图4是实施例制备的磁性FeCo双金属碳基多孔纳米酶与废纸衍生多孔碳的红外光谱图。
图5是实施例制备的磁性FeCo双金属碳基多孔纳米酶的氮气吸附-解吸图。
图6是实施例制备的磁性FeCo双金属碳基多孔纳米酶的孔尺寸分布图。
图7是实施例制备的磁性FeCo双金属碳基多孔纳米酶的X射线光电子能谱图。
图8是实施例制备的磁性FeCo双金属碳基多孔纳米酶对TMB的稳态动力学曲线图。
图9是实施例制备的磁性FeCo双金属碳基多孔纳米酶对双氧水的稳态动力学曲线图。
图10是实施例制备的磁性FeCo双金属碳基多孔纳米酶催化活性的热稳定性柱状图。
图11是实施例制备的磁性FeCo双金属碳基多孔纳米酶的类过氧化物模拟酶活性图。
图12是实施例制备的磁性FeCo双金属碳基多孔纳米酶比色分析双氧水的浓度-吸光度变化曲线图。
图13是实施例制备磁性FeCo双金属碳基多孔纳米酶比色分析血清中葡萄糖的浓度-吸光度变化曲线图,其中插图为血糖浓度与吸光度变化的线性关系图。
图14是实施例制备磁性FeCo双金属碳基多孔纳米酶比色分析葡萄糖的选择性结果图。
图15是实施例制备磁性FeCo双金属碳基多孔纳米酶比色分析谷胱甘肽(醋酸-醋酸钠缓冲体系)的浓度-吸光度变化曲线图。
图16是实施例制备磁性FeCo双金属碳基多孔纳米酶比色分析谷胱甘肽的线性曲线图,其中插图为比色分析不同浓度谷胱甘肽颜色变化的实物图。
图17是实施例制备磁性FeCo双金属碳基多孔纳米酶比色分析羊血清中谷胱甘肽的浓度-吸光度变化曲线图,其中插图为比色分析不同浓度谷胱甘肽颜色变化的实物图。
图18为本发明的制备方法流程图。
具体实施方式
以下结合附图对本发明的优选实施例进行说明,应当理解,此处所描述的优选实施例仅用于说明和解释本发明,并不用于限定本发明。
实施例
步骤1.1:将100g剪碎的碎纸屑浸泡于40ml的38wt%浓盐酸处理 24小时。
步骤1.2:将步骤1.1浸泡后的碎纸屑,洗涤、干燥。然后称取50g 进一步浸渍于50ml的7%NaOH和12%尿素(质量分数)水溶液中,然后置于-18℃冷冻24小时,解冻后得到废纸桨混合物。
步骤2.1:将10g六水合三氯化铁和8.8g六水合氯化钴完全溶解于90%乙醇溶液中,加入步骤1解冻的60g废纸桨混合物,常温搅拌2小时。
步骤2.2:将步骤2.1的产物转入水热反应釜在160℃加热,进行热聚合反应24小时。
步骤2.3:将步骤2.2热聚合反应后所得黑色沉淀物洗涤、干燥后在氮气气氛、550℃煅烧处理3小时,然后依次用蒸馏水、无水乙醇洗涤产物,最后进行干燥处理,得到磁性FeCo双金属碳基多孔纳米酶。
实验:
1.本发明磁性FeCo双金属碳基多孔纳米酶的孔结构、元素组成及酶催化动力学参数
采用物理吸附仪ASAP2020以及元素分析对实施例制备的磁性多级孔碳材料的孔结构、化学组成进行了定性、定量的详细分析;同时结合米氏方程进行实验并计算实施例1所制备磁性FeCo双金属碳基多孔纳米酶在四甲基联苯胺(TMB)和双氧水反应体系中的米氏常数(Km)和最大反应速度常数(Vmax),见表1。
表1实施例1制备的FeCo双金属碳基多孔纳米酶的质构特性、化学组成及酶催化动力学
注:表中[a]是BET表面积;[b]是总孔容量;[c]是平均介孔尺寸(BJH法)。
由表1可知,实施例1所制备磁性FeCo双金属碳基多孔纳米酶的比表面积为102.7m2/g、总孔容量为0.14cm3/g、平均介孔尺寸为5.9nm以及氧元素含量为27.3wt.%。此外,实施例1所制备FeCo双金属碳基多孔纳米酶(分别为0.27与0.043mM)对TMB和双氧水的米氏常数明显低于天然辣根过氧化物酶(0.434与3.7mM),表明FeCo双金属碳基多孔纳米酶对TMB和双氧水具有更好的亲和特性。
另外,实施例1所制备FeCo双金属碳基多孔纳米酶对TMB和双氧水的最大反应速度常数明显高于天然辣根过氧化物酶,表明FeCo双金属碳基多孔纳米酶对TMB和双氧水具有优越的催化活性(图8与图9)。不难看出,本发明方法不仅实现了变废为宝的目标,同时实现了高活性磁性碳基纳米酶的低成本制备。
此外,对实施例1所制备FeCo双金属碳基多孔纳米酶的热稳定性亦进行了研究,结果显示(图10),所制备碳基纳米酶在不同温度条件处理 2小时后的催化活性几乎无变化。然而大量研究表明,通常情况下碳基纳米酶随着温度升高天然酶的酶催化活性会显著降低。这说明本发明制备的 FeCo双金属碳基多孔纳米酶具有很好的热稳定性。
2、纳米酶活性测试
本发明采用分光光度法对实施例制备的FeCo双金属碳基多孔纳米酶的活性进行了研究。具体实验方法如下:不同浓度的FeCo双金属碳基多孔纳米酶与100μLH2O2(1.0M)、2.3mL醋酸缓冲液(200mM,pH4.6)、以及100μLTMB(10mM)混合,总体积2.5mL。按照文献所述方法进行计算,结果表明FeCo双金属碳基多孔纳米酶的活性为0.59U/mg(图11),其模拟酶活性值远高于最近报道的单原子催化剂Zn-N-C(0.03U/mg)与 Co-N-C(0.19U/mg)。
3、比色传感H2O2试验
本发明采用紫外分光光度法测试所制备FeCo双金属碳基多孔纳米酶对双氧水的分析性能。如图12所示,实验结果表明,在最佳反应条件下, 652nm处的吸光度值随双氧水浓度(5-150μM)增加逐渐增大且呈现很好的线性关系(R2=0.9967),最低检出限为0.80μM,其测定灵敏度可与大部分金属、贵金属及碳基纳米酶相媲美。
4、比色传感葡萄糖试验
本发明采用分光光度法对实施例制备的FeCo双金属碳基多孔纳米酶进行血清中葡萄糖的比色分析研究,具体试验为:首先将含不同浓度葡萄糖的健康志愿者血清100μL(加标法)与10μL葡萄糖氧化酶(5.0mgmL-1) 混合,然后加入0.89mLPBS缓冲液(0.2mM,pH7.4)并在37℃孵育60 min。紧接着将100μLFeCo双金属碳基多孔纳米酶(6.0mgmL-1),100μL TMB(8.3mM)以及1.8mLHAc-NaAc缓冲液(0.2mM,pH4.0)加入,置于45℃恒温水浴反应30min。最后进行紫外可见光谱扫描,记录652nm 处吸光度值。
实验结果表明,实施例所制备的FeCo双金属碳基多孔纳米酶对血清中葡萄糖具有很好的检测性能(见图13),随着葡萄糖浓度(10-200μM) 增加652nm处吸光度值逐渐增大其具有较好的线性关系(R2=0.9474)。值得注意的是,该方法对血糖的最低检出限为1.8μM,其检出浓度远低于健康人群(3-8mM)与糖尿病患者(9-40mM)的血糖范围。另检测灵敏度远高于目前报道的贵金属、量子点等纳米酶,说明所制备FeCo碳基纳米酶有望用于糖尿病患者的血糖监测。
为了明确实施例所制备的FeCo双金属碳基纳米酶分析血糖的选择性,选取了代表性的干扰物进行了选择性分析实验。由图14可知,血清中普遍存在的典型离子(Na+、K+与Ca2 +)、小分子化合物(酪氨酸、抗坏血酸)及糖类物质(麦芽糖、蔗糖、果糖、半乳糖)对该检测方法几乎无明显干扰现象,说明FeCo双金属碳基多孔纳米酶在血清葡萄糖分析中具有很好的选择性和应用潜力。
5、比色分析谷胱甘肽试验
为了探索实施例所制备FeCo双金属碳基多孔纳米酶在疾病标志物监测领域的应用潜能,本发明进一步建立了血清中还原型谷胱甘肽比色分析方法。由于谷胱甘肽具有清除体内自由基、调节保护蛋白、参与氨基酸的吸收与转运等多种生物功能,同时其也与肿瘤、糖尿病以及肝病等多种疾病的发病密切相关。
实验方法如下:首先制备3mL含不同浓度谷胱甘肽的缓冲溶液(0.2 M,pH=4.6)和羊血清溶液(加标法),随后依次加入100μL双氧水(200 mM)、100μLTMB(200mM)与100μLFeCo双金属碳基多孔纳米酶(0.8 mgmL-1)混合,置于40℃恒温水浴反应20min。最后扫描紫外可见光谱图,并记录652nm处的吸光度变化值(未加谷胱甘肽的分析体系为对照组)。由图15、图16可知,样品溶液在652nm处吸光度值随谷胱甘肽浓度的增大而逐渐降低,其吸光度变化值在1-30μM呈现良好的线性关系(Δ A=0.013[谷胱甘肽]-0.0086,R2=0.9948),最低检出限为0.37μM。需要指出的是,该分析方法对谷胱甘肽的灵敏度超过目前报道的大部分比色、荧光和电化学分析策略,而且随着谷胱甘肽浓度的改变分析体系的蓝色相应变化,这非常有利于实现智能手机APP分析系统的便携式监测。
另选择性实验、加标回收率实验以及羊血清实际样品分析实验(图17) 表明,目前的分析策略可实现选择性分析生物流体中谷胱甘肽的目标,说明本发明制备的FeCo双金属碳基多孔纳米酶具有很好类过氧化物模拟酶活性,有望在临床医学中替代昂贵的天然酶实现选择性、低成本、比色智能分析生物流体中H2O2、葡萄糖和谷胱甘肽。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (8)
1.一种磁性FeCo双金属碳基多孔纳米酶的制备方法,其特征在于:包括以下步骤:
步骤1:利用碎纸屑、浓盐酸、NaOH和尿素水溶液制备废纸桨混合物;具体为:
步骤1.1:将剪碎的碎纸屑浸泡于浓盐酸处理;
步骤1.2:将步骤1.1浸泡后的碎纸屑,经洗涤、干燥后进一步浸渍于NaOH和尿素水溶液中,然后置于-18℃冷冻24小时,解冻后得到废纸桨混合物;
步骤2:将六水合三氯化铁和六水合氯化钴溶解于乙醇溶液中,加入步骤1制备的废纸桨混合物,经过搅拌、热聚合反应、煅烧和洗涤后制备得到磁性FeCo双金属碳基多孔纳米酶。
2.根据权利要求1所述的一种磁性FeCo双金属碳基多孔纳米酶的制备方法,其特征在于:所述步骤2的具体步骤为:
步骤2.1:将六水合三氯化铁和六水合氯化钴完全溶解于90%的乙醇溶液中,加入步骤1制备的废纸桨混合物,常温搅拌1~4小时;
步骤2.2:将步骤2.1的产物转入水热反应釜加热,进行热聚合反应;
步骤2.3:将步骤2.2热聚合反应后的产物在惰性气体保护下,进行煅烧处理,然后依次用蒸馏水、无水乙醇洗涤产物,最后进行干燥处理,得到磁性FeCo双金属碳基多孔纳米酶。
3.根据权利要求2所述的一种磁性FeCo双金属碳基多孔纳米酶的制备方法,其特征在于:所述步骤1.1的浓盐酸为38wt%,处理时间为24小时,其中碎纸屑和浓盐酸的配比为5g:2ml。
4.根据权利要求3所述的一种磁性FeCo双金属碳基多孔纳米酶的制备方法,其特征在于:所述步骤1.2的NaOH溶液质量分数为7%,尿素溶液质量分数为12%,其中处理后的碎纸屑与NaOH和尿素混合水溶液的比例为1g:1ml。
5.根据权利要求4所述的一种磁性FeCo双金属碳基多孔纳米酶的制备方法,其特征在于:所述步骤2.1中,六水合三氯化铁和六水合氯化钴中Fe(III)与Co(II)的摩尔比为1.67。
6.根据权利要求5所述的一种磁性FeCo双金属碳基多孔纳米酶的制备方法,其特征在于:所述步骤2.2中水热反应釜温度为160~180℃,热聚合反应时间为12~24小时。
7.根据权利要求6所述的一种磁性FeCo双金属碳基多孔纳米酶的制备方法,其特征在于:所述步骤2.3的煅烧的温度为550℃,时间为3小时。
8.权利要求1~7任意一项制备方法制备的磁性FeCo双金属碳基多孔纳米酶的应用,其特征在于:选择性比色传感生物流体中H2O2、葡萄糖和谷胱甘肽。
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