CN103255177B - 一种生物还原制备氮硫同时掺杂石墨烯的方法 - Google Patents
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Abstract
本发明提供了一种生物还原制备氮硫同时掺杂石墨烯的方法，属于石墨烯的合成和纳米材料技术领域。该方法以氧化石墨烯分散溶液为原料，以含硫酸盐还原菌菌悬液作为生物还原剂，在控制反应体系中隔绝空气条件下反应5-10天得到石墨烯溶液，离心过滤和反复洗涤后即得到氮、硫掺杂的石墨烯。本发明方法不添加任何稳定剂或分散剂，利用微生物的代谢活动还原氧化石墨烯得到氮硫掺杂的石墨烯，具有安全无毒、操作简单、绿色环保等优点，解决了现有的石墨烯制备方法中难于将氮、硫同时掺杂的重大难题，氮硫同时掺杂石墨烯应用于电化学传感器和电催化等领域时可显著提高检测限和检测灵敏度，应用前景广阔。
Description
技术领域
本发明涉及一种氮、硫同时掺杂的石墨烯的制备方法，属于石墨烯的合成和纳米材料技术领域，具体是一种基于硫酸盐还原菌(SRB)还原制备石墨烯的方法。
背景技术
石墨烯(Graphene)是一种独特的二维平面结构单层碳纳米材料，碳原子之间以共价键连结形成六方蜂巢结构。石墨烯的这一独特的结构特点使其具有良好的电学、力学和热学性质，近年来在微电子，新型超导材料、能量储存、传感器以及电催化等方面具有广阔的应用前景。而同时围绕石墨烯的基础与应用研究也在世界范围内如火如荼的展开了。
石墨烯制备方法的研究是其基础与应用研究的前提。目前文献报道的主要有机械剥离、外延生长、化学气相沉积和化学还原法。这些方法各有优势，同时也有各自的缺点。前三种方法因生产成本高、操作工艺复杂、实验条件苛刻而难以应用到石墨烯的大规模生产中。相比较而言，化学还原氧化石墨烯法是当前大规模制备石墨烯的常用方法。然而因广泛采用水合肼、二甲基肼、多巴胺、硼氢化钠等有毒还原剂，在一定程度会造成环境污染、危害健康，因此探索出一种简单、经济、绿色环保的还原工艺和方法是一个亟待解决的问题。
硫酸盐还原菌是一类形态各异、营养类型多样、能利用硫酸盐或者其它氧化态硫化物作为电子受体来异化有机物质的微生物。广泛存在于土壤、水田、湖、沼、河川底泥、海底泥、石油矿床、温泉水和地热地区，甚至于存在人体的口腔和肠道中。在富含硫酸盐的有水厌氧环境中，硫酸盐还原菌是相当活跃的分子，它以硫酸盐做最终电子受体，分解水体中的有机物，获得合成细胞物质和维持生命所需的能量。硫酸盐作为硫酸盐还原菌代谢过程中的最终电子受体，将还原成硫离子，它首先在细胞体外积累，然后进入细胞，在细胞内，第一步反应是SO4 2-的活化，即SO4 2-与ATP反应转化为腺苷酰硫酸(APS)和焦磷酸(PPi)，PPi很快分解为无机磷酸(Pi)，推动反应不断向左进行。APS继续分解成亚硫酸盐和磷酸腺苷(AMP)。亚硫酸盐脱水后变成偏亚硫酸盐(S2O5 2-)，S2O5 2-极不稳定，很快转化为中间产物连二亚硫酸盐(S2O4 2-)，S2O4 2-又迅速转化为S3O6 2-，S3O6 2-分解成硫代硫酸盐(S2O3 2-)和亚硫酸盐(SO3 2-)，S2O3 2-又经自身的氧化还原作用，变成SO3 2-和最终产物S2-，S2-被排出体外，进入周围环境。有关反应方程如下：
硫酸盐还原菌的生物还原作用是本发明的关键，即利用细菌生物还原酶的作用，在自然温和的条件下还原制备石墨烯。此外，由于生物体本身还有氮、磷等各种微量元素，因此在石墨烯的元素掺杂方面可能存在更多的惊喜。
发明内容
为解决上述石墨烯制备过程中出现的各种技术缺陷，本发明旨在提供一种生物催化还原氧化石墨烯，得到氮、硫同时掺杂石墨烯的方法。本方法简单、经济、绿色环保。
实现本发明的技术方案是：
本发明提供的生物还原制备氮硫同时掺杂石墨烯的方法，包括以下步骤：
(1)将氧化石墨烯分散在水中，浓度为0.05mg/ml～0.5mg/ml；
(2)将硫酸盐还原菌(SRB)进行活化培养；
(3)硫酸盐还原菌(SRB)培养基采用高压蒸汽灭菌；
(4)将上述三个步骤分别得到的三种溶液混合密封，保证不存在空气，在37℃恒温培养箱中培养5～7天；
(5)离心过滤和反复洗涤后即得到氮、硫掺杂的石墨烯。
上述方法中，硫酸盐还原菌(SRB)菌液与硫酸盐还原菌(SRB)培养基的体积比例为1∶1～1∶5；硫酸盐还原菌(SRB)菌液与氧化石墨烯的体积比例为1∶2～1∶10。
在上述方法的步骤(1)中，将氧化石墨烯分散在水中的同时施加超声分散；在步骤(2)中，将硫酸盐还原菌(SRB)进行活化培养是采用不添加维生素C和硫酸亚铁铵的API培养基培养2～3次，所得硫酸盐还原菌(SRB)菌液为浅黄色浊液。硫酸盐还原菌(SRB)培养基的组成是：0.5％～0.2wt％的NaCl，0.05wt％～1wt％的MgSO4·7H2O，0.01wt％～0.5wt％的酵母浸汁液，0.005wt％～0.2wt％的K2HPO4。
上述方法中所述的硫酸盐还原菌(SRB)是自国内某油田采出水中分离培养，经多代驯化纯化后的菌种，该菌种具有生物还原酶，其生长适宜的温度为30～50℃，生长周期为7～15天。
本发明方法以氧化石墨烯分散溶液为原料，以含硫酸盐还原菌菌悬液作为生物还原剂，含硫酸盐还原菌菌悬液是由硫酸盐还原菌菌种和硫酸盐还原菌培养基组成，并控制反应体系中隔绝空气，在一定温度下反应5-10天后，得到石墨烯溶液，离心过滤和反复洗涤后即得到氮、硫掺杂的石墨烯。所述的氧化石墨烯分散溶液分散在超纯水中，浓度为0.05mg/ml～0.5mg/ml，溶于水的同时超声分散。
本发明提供了一种利用微生物还原制备氮硫掺杂石墨烯的方法，属于石墨烯的合成和纳米材料技术领域。所述石墨烯可用来开发用于纳米电子学、纳米复合物、纳米电化学传感器、电催化、氢贮藏以及生物应用的石墨烯纸或石墨烯薄膜、基于石墨烯的复合物以及制品。本发明的制备方法是在硫酸盐还原菌(SRB)菌悬液中，不添加任何稳定剂或分散剂，利用微生物的代谢活动还原氧化石墨烯得到氮、硫同时掺杂的石墨烯。本发明所提供的方法无需采用毒性强的水合肼、硼氢化钠等化学还原剂，具有操作简单、绿色环保等优点；氮硫同时掺杂的石墨烯可解决现有的石墨烯制备方法中难于将氮、硫同时掺杂的重大难题，应用于电化学传感器和电催化等领域时可显著提高检测限和检测灵敏度，应用前景十分广阔。本发明的创新点主要在于以下两个方面：(1)硫酸盐还原菌分布广，培养简便，利用生物还原酶制备石墨烯工艺简单，条件温和且环境友好，适合低成本、大规模、绿色生产需要。(2)生物制备的石墨烯为氮硫同时掺杂，应用于电化学传感器和电催化等领域时可显著提高检测限和检测灵敏度。
附图说明
图1是本发明所述氧化石墨烯荣格和石墨烯溶液的紫外光谱图。其中，图a是氧化石墨烯的紫外光谱图；图b是SRB还原制备的石墨烯的紫外光谱图。
图2是本发明所述的氧化石墨烯和石墨烯的X射线衍射(XRD)图。其中，图a是氧化石墨烯的XRD图；图b是SRB还原制备的石墨烯的XRB图。
图3是本发明所述氧化石墨烯的X光电子能谱(XPS)图。其中，图a是氧化石墨烯的XPS图；图b是SRB还原制备的石墨烯的XPS图；图c是SRB还原制备石墨烯中的N1s谱图；图d是是SRB还原制备石墨烯中的S2p谱图。
图4是本发明所述的生物还原制备氮硫同时掺杂的石墨烯以及未经掺杂的石墨烯、仅氮掺杂石墨烯分别修饰玻碳电极后，采用差分脉冲伏安法检测溶液中Pb和Cd的离子浓度谱图。
具体实施方式
实施例1氧化石墨烯的制备
将1.5g天然鳞片石墨粉与1.5g硝酸钠和69ml浓硫酸在冰浴中混合均匀。缓慢加入9g高锰酸钾，在常温水浴下搅拌反应1h。逐渐加入100ml去离子水，温度逐渐升高，在90℃下搅拌继续反应30min，混合物由棕褐色变成亮黄色，加入10ml，30％双氧水溶液，混合物由棕褐色变成亮黄色。趁热过滤，采用盐酸溶液和去离子水充分洗涤至滤液呈pH＝6。将氧化石墨烯滤饼在80℃真空烘箱中充分干燥，保存备用。
实施例2 SRB菌种的活化
取出冰箱中保存的SRB菌种，置于适宜温度下的恒温培养箱中保温2～3小时，移取适量的菌种于已灭菌的瓶子中，将已灭菌培养基加满整个瓶子，盖上塞子，保证瓶中没有空气。将接种有SRB的培养液置于恒温培养箱中培养24小时，如果瓶中溶液变黑，则说明所培养的SRB具有相当的活性。如果没有变黑，那么就按上述操作步骤重新接种一次，直到瓶中的溶液变黑，如果接种三次瓶中的溶液都没有变黑，那么说明菌种已经失活，需要更换菌种。
实施例3 石墨烯修饰电极检测重金属离子
(1)通过Hummers法制得氧化石墨烯。将氧化石墨烯分散在水中，并进行超声分散，制成0.1mg/ml的分散液。
(2)SRB培养基组成如下：乳酸钠4.0ml；酵母浸汁液1.0g；七水硫酸镁0.2g；磷酸氢二钾0.01g；氯化钠10g；蒸馏水1000ml。用氢氧化钠调pH至7.0-7.2，在0.12×105Pa的高压蒸汽下，灭菌20min，快速冷却至37℃左右，恒温保存备用。
(3)SRB菌株来自我国胜利油田污水。按应用实例2的方法活化后备用。
(4)取100ml浓度为0.1mg/ml的氧化石墨烯溶液、20ml稀释培养所得的具有相当活性的SRB菌液和30ml新鲜培养基混合均匀，密封保存，以保证液面上部没有空气。然后置于37℃的恒温培养箱中培养7天。对所制备的石墨烯溶液进行紫外光谱(图1b)表征。
(5)将黑色悬浊液离心(14000rpm)分离，然后加入1mol/LHCl和超纯水充分洗涤至滤液呈中性。最后将离心所得的下层沉淀物置于80℃真空干燥箱中充分干燥，得到氮、硫同时掺杂单层石墨烯。对所制备的氮、硫掺杂石墨烯进行X射线衍射(图2b)和X光电子能谱(图3b，3c，3d)表征。
实施例4 采用石墨烯修饰电极电化学检测溶液中Pb和Cd的浓度
(1)玻碳电极预处理
将直径3mm的玻碳(GC)电极先在1200#金相砂纸上打磨，然后依次在1.0，0.3，0.05μm三氧化二铝悬浊液中抛光成镜面，再依次在1∶1乙醇、1∶1硝酸和超纯水中超声清洗3次(约5min/次)，然后在空气中自然晾干备用。
(2)修饰电极制备
用吸量管分别取2.00ml还原性石墨烯悬浊液和2.00ml0.1％Nafion-乙醇溶液混合超声分散15min，配制成0.25mg/ml Graphene-Nafion混合液，然后采用微量注射器吸取5μl浊液滴涂在玻碳电极镜面上(尽量让浊液在表面分布均匀)，放入红外快速干燥箱快速干燥5min后，取出待用。
(3)电化学检测
采用N，S-G/Nafion/GC电极，辅助电极为铂丝电极，参比电极为饱和甘汞电极，以0.2mol/L醋酸盐缓冲溶液(pH＝4.6)为测试底液。于电解池中加40mL0.2mol/L HAc-NaAc缓冲溶液(pH＝4.6)，再加入适量的Pb(II)和Cd(II)标准溶液，插入三电极体系，设定沉积电位为-1.2V，沉积时间为300s(搅拌)，预富集结束后停止搅拌，静止10s，采用差分脉冲伏安法由负向正扫描，测量并记录溶出曲线。每次测定结束后，调节工作电极在+0.3V下清洗120s。清洗后的电极可重复使用。实验无需通氮除氧，均在室温条件下进行。由图4中可看出，本发明制备的氮硫同时掺杂石墨烯修饰电极大大提高了Pb和Cd离子的检测限。
Claims (6)
1.一种生物还原制备氮硫同时掺杂石墨烯的方法，包括以下步骤：
(1)将氧化石墨烯分散在水中，浓度为0.05mg/ml～0.5mg/ml；
(2)将硫酸盐还原菌进行活化培养；
(3)硫酸盐还原菌培养基采用高压蒸汽灭菌；
(4)将上述三个步骤分别得到的三种溶液混合密封，保证不存在空气，在37℃恒温培养箱中培养5～7天；
(5)离心过滤和反复洗涤后即得到氮、硫掺杂的石墨烯。
2.根据权利要求1所述的生物还原制备氮硫同时掺杂石墨烯的方法，其特征在于，硫酸盐还原菌菌液与硫酸盐还原菌培养基的体积比例为1:1～1:5。
3.根据权利要求1所述的生物还原制备氮硫同时掺杂石墨烯的方法，其特征在于，硫酸盐还原菌菌液与氧化石墨烯分散液的体积比例为1:2～1:10。
4.根据权利要求1所述的生物还原制备氮硫同时掺杂石墨烯的方法，其特征在于，在步骤(1)中，将氧化石墨烯分散在水中的同时施加超声分散。
5.根据权利要求1所述的生物还原制备氮硫同时掺杂石墨烯的方法，其特征在于，在步骤(2)中，将硫酸盐还原菌进行活化培养是采用不添加维生素C和硫酸亚铁铵的API培养基培养2～3次，所得硫酸盐还原菌菌液为浅黄色浊液。
6.根据权利要求1所述的生物还原制备氮硫同时掺杂石墨烯的方法，其特征在于，硫酸盐还原菌培养基的组成是：0.5％～0.2wt％的NaCl，0.05wt％～1wt％的MgSO4·7H2O，0.01wt％～0.5wt％的酵母浸汁液，0.005wt％～0.2wt％的K2HPO4。
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