CN113186229A - 一种光热金属纳米材料硒化铜的快速可控生物合成方法 - Google Patents
一种光热金属纳米材料硒化铜的快速可控生物合成方法 Download PDFInfo
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Abstract
本发明涉及光热金属纳米材料合成技术领域,公开了一种光热金属纳米材料硒化铜的快速可控生物合成方法。本发明方法中反应体系的Cu2‑xSe纳米颗粒形成过程无需依赖特定胞内或胞外还原酶,而只需要将细菌胞内代谢产生的电子通过细胞膜上的电子传递链连续不断的转移至胞外,并通过溶解性电子媒介进一步实现Na2SeO3后与Cu2+的胞外还原以及Cu2‑xSe纳米颗粒自组装。因此,该体系的Cu2‑xSe纳米颗粒生物合成机理显著区别于传统方法。
Description
技术领域
本发明涉及光热金属纳米材料合成技术领域,具体的说是涉及一种光热金属纳米材料硒化铜的快速可控生物合成方法。
背景技术
硒化铜(Cu2-xSe)纳米颗粒是一种具有独特的光电、光热性质的金属纳米材料,被广泛应用于热电器件制造、催化和医学治疗等领域。目前,Cu2-xSe纳米材料的大规模制备主要采用化学合成方法,存在能耗与成本高(需高温高压反应条件)、操作复杂、产生二次污染等问题。近年来,基于微生物代谢过程的生物合成方法作为一种绿色、简便的纳米材料制备方法开始受到广泛关注。但是,微生物复杂的胞内代谢途径会干扰金属的生物转化和金属纳米材料的生物合成过程,并且有毒金属离子进入胞内会降低细胞代谢活性从而抑制材料的生物合成。因此,现有的生合成体系难以实现纳米材料高效、可控的合成,并且所合成的纳米材料在质量和性能上往往无法与化学合成的同类产品相媲美。
目前,利用生物合成方法制备硒化物的方法普遍采用胞内合成途径,具体途径为:利用胞内特定的硒还原酶、还原性巯基蛋白等将(亚)硒酸盐还原生成低价态的有机硒,进而利用胞内的巯基蛋白或植物螯合肽等抓捕进入胞内的重金属离子,两者在胞内再而反应形成硒化物。然而,由于胞内代谢途径与产物的复杂,一部分进入胞内的重金属会生成硫化物、磷化物、氢氧化物等副产物,从而显著降低了纳米材料产品的纯度和合成效率。目前,关于Cu2-xSe纳米颗粒的生物合成仅有两篇论文报导。其中,有研究利用一株硒还原菌Pantoeaagglomerans同时还原Cu2+和SeO3 2-,在Pantoea agglomerans体系中,细胞分泌少量氧化还原蛋白至胞外,进而还原溶液中的Na2SeO3后与Cu2+反应生成Cu2-xSe纳米材料。然而,该体系材料合成过程中SeO3 2-的还原依赖有限的胞外酶,还原能力弱且无法持续还原(仅能还原约0.1mM Cu2+和SeO3 2-),并且制备的Cu2-xSe缺少产量、光热性能等信息,因此难以判断该合成方法的实际应用效果。
另外,有研究利用Shewanella oneidensis合成了较高光热性能的Cu2-xSe纳米颗粒,但细胞仍然主要依靠胞内的功能酶还原Cu2+和Na2SeO3,产率仍然较低,并且在胞内形成Cu2-xSe纳米颗粒,因此生物合成过程仍受到细胞毒性和胞内代谢的影响,产品性能仍有待提高。因此,现有生物合成方法尚难以实现Cu2-xSe材料的大规模、快速可控制备。
发明内容
有鉴于此,本发明的目的在于提供一种光热金属纳米材料Cu2-xSe的快速可控生物合成方法,使得所述合成方法能够高效率、快速和可控的合成光热金属纳米材料Cu2-xSe。
为了实现上述目的,本发明提供如下技术方案:
一种光热金属纳米材料硒化铜的快速可控生物合成方法,包括:
步骤1、将异化金属还原细菌在好氧条件下活化;
步骤2、将活化后的异化金属还原细菌接种至厌氧培养基中,然后加入溶解性电子媒介、二价铜盐和亚硒酸盐,在厌氧条件下反应,通过差速离心将菌体分离在上清液中,收集沉淀物,获得Cu2-xSe纳米颗粒;其中,x=0-1。
Cu2-xSe(硒化铜)中的2-x中的x表示材料的铜元素(包含一价和二价铜)中二价铜的占比。依据生物合成机制,细菌初始合成的纳米材料为Cu2Se,但因Cu2Se材料中的一价铜易转换成二价形成铜缺陷。因此,本领域中铜缺乏的硒化铜材料通常被表示为Cu2-xSe(x=0-1)。虽然通过添加AQDS实现胞外还原Na2SeO3的方法已有报导,但Cu2-xSe纳米颗粒的胞外合成还需同时实现Cu2+还原生成Cu+进而与Se还原产物作用生成Cu2-xSe。目前,尚未有利用溶解性氧化还原活性物质来促进微生物胞外还原Cu2+生成Cu+的报导,更缺少利用该方法调控Cu2-xSe纳米颗粒生物合成的研究。
因此,本发明充分利用Shewanella oneidensis等异化金属还原菌的胞外电子传递能力,提出通过添加/自合成氧化还原活性物质(如蒽醌-2,6-二磺酸钠AQDS、核黄素RF等)介导Cu2-xSe纳米颗粒胞外合成的新方法。
作为优选,步骤1为:
预先接种活性良好的电化学活性菌并在好氧条件下培养12h,然后将菌液转接至新鲜的LB培养液中持续好氧培养12h。
作为优选,所述溶解性电子媒介在厌氧培养基中的浓度不高于对异化金属还原细菌生长有毒性的浓度。优选的,其安全浓度为0-200μM,在本发明具体实施方式中,所述溶解性电子媒介浓度选择100μM进行试验。
作为优选,所述厌氧培养基为LB培养基或异化金属还原细菌的矿物盐培养基,pH值为6.8-7.2。实际使用中,曝氮气后灭菌使用,曝气时长为20-30分钟。
作为优选,所述异化金属还原细菌为希瓦氏菌和/或大肠杆菌。更优选地,所述希瓦氏菌为奥奈达希瓦氏菌(Shewanella oneidensis),如Shewanella oneidensis MR-1和Shewanella putrefaciens CN32,而大肠杆菌可以选择大肠杆菌Jm109,这些菌株具备胞外电子传递还原AQDS和RF的能力。
作为优选,所述希瓦氏菌是能够自身合成溶解性电子媒介的工程菌。在本发明具体实施方式中,本发明充分利用希瓦氏菌可以自合成氧化还原活性物质核黄素(RF)的特性,进一步提出并证明了利用微生物自合成的RF来加速Cu2-xSe生物合成的新方法。基于此构建了过表达RF合成基因质粒的基因工程菌株,该菌株同样具有快速合成Cu2-xSe纳米颗粒的能力并且无需外加氧化还原活性物质,从而进一步提高了该方法的可持续和环境友好性。该菌株可按照常规基因工程技术对进行Shewanella oneidensis改造制备,如从黄素合成基因簇ribD-ribC-ribBA-ribE中扩增序列,经双酶切和纯化后,克隆到表达质粒中。将拟引入Shewanella oneidensis的表达质粒首先转化到质粒供体菌株上,通过偶联法导入Shewanella oneidensis。
作为优选,所述二价铜盐选自氯化铜、硫酸铜等水溶性二价铜盐,所述亚硒酸盐为亚硒酸钠等水溶性亚硒酸盐。
作为优选,所述厌氧条件下反应的时间为9-24h。反应充分恒温(30-32℃)振荡(200-300rpm)培养。
与现有生物合成Cu2-xSe纳米颗粒相比,本发明具有如下优点(参见表1):
表1
(1)本发明实施例采用的菌株为希瓦氏菌和大肠杆菌,相对于其他菌株,具有生长速度快、向胞外传递电子(持续还原力)、遗传操作简便等诸多优点,因此具有成熟的大规模培养工艺。本发明可用菌株不限于具体实施例所用菌株,所有具有胞外电子传递能力的异化金属还原菌均可用于合成本发明所述Cu2-xSe纳米材料。
(2)本发明提出投加溶解性电子媒介AQDS和RF将生物Cu2-xSe的合成场所由胞内转移至胞外的方法,降低了投加前驱物对细菌的毒性作用,从而使细菌能维持高活性代谢水平,实现Cu2-xSe纳米颗粒的高效、快速合成。利用本方法,可获得高性能、高纯度的Cu2-xSe纳米颗粒,并将原有体系合成的周期由约7天缩短至仅需4-9小时内,具有巨大的性能优势和实际应用潜力。
附图说明
图1所示为实施例1中调控生物Cu2-xSe纳米颗粒合成过程中的细菌切片TEM图;
图2所示为实施例1中电子媒介调控下快速合成生物Cu2-xSe纳米颗粒浓度数据;WT表示shewanella oneidensis MR-1野生型细菌;
图3所示为实施例1中电子媒介调控下生物Cu2-xSe纳米颗粒XRD图;
图4所示为实施例2中基因工程菌株调控快速合成Cu2-xSe纳米颗粒浓度数据图;WT表示shewanella oneidensis MR-1野生型细菌;
图5所示为实施例3中E.Coli Jm109合成Cu2-xSe纳米颗粒的TEM-EDX图。
具体实施方式
本发明实施例公开了一种光热金属纳米材料硒化铜的快速可控生物合成方法,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明内。本发明所述合成方法已通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的合成方法进行改动或适当变更与组合,来实现和应用本发明技术。
本发明反应体系的Cu2-xSe纳米颗粒形成过程无需依赖特定胞内或胞外还原酶,而只需要将细菌胞内代谢产生的电子通过细胞膜上的电子传递链连续不断的转移至胞外,并通过溶解性电子媒介进一步实现Na2SeO3后与Cu2+的胞外还原以及Cu2-xSe纳米颗粒自组装。因此,该体系的Cu2-xSe纳米颗粒生物合成机理显著区别于传统方法。现有生物合成体系都不具备或未利用细菌的胞外电子传递能力,而本发明通过往Shewanella oneidensis和Jm109培养液中加入适量的溶解性电子媒介强化胞外电子传递过程,使Cu2-xSe纳米颗粒生物合成的场所从胞内转移到胞外,从而减轻了高浓度前驱物对细胞的毒性和胞内反应的干扰,保证了细菌的高电子输出能力,进而促进了Cu2-xSe纳米颗粒的高效、快速、可控生物合成。
以下就本发明所提供的一种光热金属纳米材料硒化铜的快速可控生物合成方法做进一步说明。
实施例1:以奥奈达希瓦氏菌(shewanella oneidensis MR-1)合成Cu2-xSe纳米颗粒
(1)希瓦氏菌的培养:选取菌种奥奈达希瓦氏菌(shewanella oneidensis MR-1);向50mL的厌氧LB培养基(含酵母提取物5g/L,胰蛋白胨10g/L和氯化钠10g/L,pH=7)中接入希瓦氏菌种,于30℃恒温振荡(200rpm)12小时获得菌液;以体积比例为1:10将菌液转移至200mL的LB培养基中在相同条件下继续活化12h后获得菌液;
(2)Cu2-xSe的快速合成:将获得的菌液离心收集,用LB培养基洗2-3遍后重悬,将细菌悬浮液(OD600=3)转移至步骤(1)中的厌氧LB体系中。将100μM水溶性的AQDS/RF加入到厌氧体系,之后依次加入0.3mM水溶性亚硒酸盐与0.6mM的水溶性二价铜盐。30℃恒温振荡培养,转速为200rpm,培养时间为9-48h,获得Cu2-xSe材料;
(3)Cu2-xSe材料的回收:生物合成反应48h后,将菌液离心1-2min(4000-5000rpm),去除上清液,将收集到的固体沉淀置于冷冻干燥机中干燥。使用玛瑙研钵将干燥后的固体研磨成均匀的粉末,取适量粉末进行X射线衍射分析表征。
对本实施例所快速制备的生物合成Cu2-xSe纳米材料(标记为①)进行性能指标测试:
(1)细菌切片及TEM表征样品制备:取适量步骤(2)合成后的溶液,6000g离心5min,弃上清。用2.5%的戊二醛与4%的多聚甲醛重悬沉淀,固定12h。用PBS冲洗3遍,采用浓度梯度的乙醇进行脱水并用环氧树脂包裹,切为50-100nm厚度的纳米片,并置于铝网上进行透射电子显微镜表征。图1为实施例1不同时间段生物合成的Cu2-xSe纳米材料的TEM图。由图可见在AQDS存在时,Cu2-xSe的合成速率明显快于无AQDS组,并且Cu2-xSe呈球形颗粒均匀分布在胞外,无团聚,粒径为50-80nm。
(2)生物转化的Se与Cu元素浓度ICP-AES测试:取适量步骤(2)合成后的溶液,9000g离心5min,弃上清,用超纯水重悬沉淀并清洗4-5遍。将获得的沉淀进行消解,加入4mL硝酸高温煮沸,后加入1mL高氯酸,当消解管冒浓白烟时消解结束,将溶液体积定容5mL,采用ICP-AES测量溶液中Se与Cu元素浓度。图2为合成过程中已被生物转化的Se与Cu元素浓度,由图可知,AQDS/RF存在时,9h已经基本完成Cu2-xSe的合成,而24h已经完成合成。
(3)XRD表征样品制备:图3为本实施例采用AQDS合成的Cu2-xSe的X射线衍射分析表征,通过对比标准卡片(#21-1016),说明实施例1中合成的纳米材料物相为Cu5Se4(Cu1.25Se),x=0.75,即Cu:Se的摩尔比值为1:1.25。
实施例2:过表达核黄素合成基因的奥奈达希瓦氏菌(shewanella oneidensisMR-1)合成Cu2-xSe纳米颗粒
本发明还构建了利用过表达核黄素合成基因的pYYDT-Rib基因工程菌株快速合成Cu2-xSe纳米颗粒的新体系。
从黄素合成基因簇ribD-ribC-ribBA-ribE中扩增序列,经SpeI和Sbf I纯化后,克隆到pYYDT表达质粒中,形成pYYDT-Rib表达质粒。将拟引入奥尼氏球菌MR-1的pYYDT-rib质粒首先转化到质粒供体菌株E.Coli WM3064上,通过偶联法导入S.oneidensis MR-1。
合成方法与实施例1基本相同,仅将合成材料的细菌更改为过表达核黄素RF合成基因的pYYDT-Rib(MR-1)菌株,在培养细菌的体系投加50μg/mL的卡那霉素以及在厌氧合成体系投加终浓度为10mM的阿拉伯糖诱导核黄素合成基因的过表达(这些操作均取决于工程菌所带的诱导表达系统和筛选标签,可以实际情形调整)。将pYYDT-Rib菌株合成纳米材料标记为②;对本实例中制备的纳米颗粒进行ICP-AES的浓度表征。
图4的ICP-AES的结果表明Cu:Se的摩尔比值为1.25,即本发明同样合成了Cu2-xSe纳米材料,其对应的数据见下表2;
表2
实施例3:E.Coli Jm109菌株合成Cu2-xSe纳米颗粒
本发明还构建了利用E.Coli Jm109菌株快速合成Cu2-xSe纳米颗粒的新体系。
操作步骤与实施例1基本相同,仅将合成材料的细菌更改为菌株E.Coli Jm109,将细菌的培养温度更改为E.Coli Jm109的适应温度36-38℃。将Jm109菌株合成纳米材料标记为③;对本实例中制备的纳米颗粒进行TEM-EDX表征,结果如图5。
根据图5可见纳米颗粒分布在胞外,并且TEM-EDX结果说明合成的为Cu2-xSe纳米颗粒。通过ICP-AES测试的Cu:Se的原子摩尔比为1.74,即获得了x=0.26的Cu1.74Se纳米颗粒。
以上所述只是用于理解本发明的方法及其核心思想,应当指出,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下,可以对本发明进行若干改进和修饰,这些改进和修饰也落入本发明权利的保护范围。
Claims (10)
1.一种光热金属纳米材料硒化铜的快速可控生物合成方法,其特征在于,包括:
步骤1、将异化金属还原细菌在好氧条件下活化;
步骤2、将活化后的异化金属还原细菌接种至厌氧培养基中,然后加入溶解性电子媒介、二价铜盐和亚硒酸盐,在厌氧条件下反应,通过差速离心将菌体分离在上清液中,收集沉淀物,获得Cu2-xSe纳米颗粒;其中,x=0-1。
2.根据权利要求1所述合成方法,其特征在于,步骤1为:
预先接种活性良好的异化金属还原细菌并在好氧条件下培养12h,然后将菌液转接至新鲜的LB培养液中持续好氧培养12h。
3.根据权利要求1所述合成方法,其特征在于,所述溶解性电子媒介在厌氧培养基中的浓度不高于对异化金属还原细菌生长有毒性的浓度。
4.根据权利要求1或3所述合成方法,其特征在于,所述厌氧培养基为LB培养基或异化金属还原细菌的矿物盐培养基。
5.根据权利要求1、2或3所述合成方法,其特征在于,所述异化金属还原细菌为希瓦氏菌和/或大肠杆菌。
6.根据权利要求5所述合成方法,其特征在于,所述希瓦氏菌是能够自身合成溶解性电子媒介的工程菌。
7.根据权利要求1、3或6所述合成方法,其特征在于,所述溶解性电子媒介为蒽醌-2,6-二磺酸钠和/或核黄素。
8.根据权利要求1所述合成方法,其特征在于,所述二价铜盐为氯化铜和/或硫酸铜。
9.根据权利要求1所述合成方法,其特征在于,所述亚硒酸盐为亚硒酸钠。
10.根据权利要求1所述合成方法,其特征在于,所述厌氧条件下反应的时间为9-24h。
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