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生物动态自组装在自然界中普遍存在，并构成了生物纳米机器以及很多自然材料系统的结构和功能基础。如骨和贝壳等自然材料系统就是在细胞的参与下，对有机和无机组分进行时空可控的动态自组装而形成。这些自然材料体系具备无与伦比的结构多级性，很多材料性能（比如机械性能）也远优于人工合成的材料。此外，这些动态自组装体系还具有过程自适应、环境响应和自我修复等特质。因此，将自然动态自组装体系整合嵌入合成自组装体系代表着一种新的自组装方法，该方法在创造新的材料和纳米结构方面蕴含着极大潜力，而这方面的交叉研究基本属于空白。
上海科技大学物质学院材料与物理生物学研究部钟超教授课题组利用前沿合成生物学技术，研发了一种经过基因编程改造的智能细菌。该细菌能响应环境比如光和小分子，并能像经过高精度编程的计算机程序一样，按人为预先设定的方式进行工作。在这项研究中，经基因编程的细菌，能够时空可控地对溶液中的无机纳米材料进行动态自组装。近日，该研究成果发表在国际知名学术期刊《Advanced Materials》上: Xinyu Wang et al., Programming Cells for Dynamic Assembly of Inorganic Nano-objects with Spatiotemporal Control, Adv. Mater.， 2018, 1705968。该论文的创新性和研究成果备受审稿人和编辑的肯定和推崇，不仅入选2018年4月第16期内封面文章，还被《Advanced Materials》编辑推荐在官方网站的视频摘要(video abstract)中进行重点介绍。 在这项工作中，为了挖掘自然动态自组装体系蕴含的潜力，实现其和合成的无机纳米材料的完美整合，钟超课题组利用合成生物学技术，对大肠杆菌的生物被膜淀粉样蛋白基因进行了改造。大肠杆菌生物被膜的主要成分是卷曲纤毛纤维，其主要成分是通过大肠杆菌分泌的CsgA蛋白亚基自组装而成。首先，课题组通过对大肠杆菌生物被膜CsgA蛋白分泌基因的改造，开发出光控（蓝光）调控CsgA蛋白表达和分泌的基因环路；其二，通过对CsgA蛋白进行功能修饰，能让分泌的CsgA蛋白自动识别经有机小分子配体修饰的无机纳米材料。在光的诱导下，工程菌能吐出大量的CsgA功能蛋白，并在细胞周围自组装形成纳米纤维材料网络，由于生成的纳米纤维在很多界面都具有超强的粘附作用，因而细菌最终能对溶液中的无机纳米材料进行大规模、多尺度并按时空可控的方式在各种界面进行动态自组装。
该研究首先证明了单种和多种纳米颗粒在不同基底表面的动态、大规模、多尺度组装。蓝光光控基因线路的引入则可以控制纳米颗粒的空间布阵，布阵精度可达100μm。另外，通过控制纳米颗粒的添加顺序，还可以实现纳米物件的自动层层自组装。该团队提出的此项动态纳米物件自组装方法，在生物电子，光电器件，生物催化和可穿戴设备方面都具有潜在的应用价值。在示例应用当中，他们利用导电生物被膜制备了叉指电极阵列，并证明其可作为触碰开关装置。该纳米材料动态自组装方法还可用于创造活体功能材料，将无机体系的光电高效性和自然活体体系催化的高选择性结合起来，应用于人工光合作用体系。同时，本项研究也为合成生物学在材料和生物纳米技术上的应用提供了一个很好的范例。 Figure 1. Schematic of the designer cell-enabled strategy for diverse and complex assembly of inorganic nano-objects (Ns) in adynamic, scalable, and hierarchical fashion through programmable dynamic biofilm formation. a) The designer cell-enabled strategy resides on the integration and synchronization of templated assembly of NOs with programmable E. coli curli fiber assembly regulated by synthetic gene circuits, induced by chemical (tetracycline) or blue light (470 nm). Upon exposure to tetracyclineor blue light, E. coli cells express CsgAHis (major subunit of curli), which are then secreted and assembled into curli fibers associated with cell membrane. Dynamic assembly of NTA-decorated NOs on curli fibers comprising CsgAHis proteins was enabled by co-incubation in M63 media based on“NTA–Metal–His” coordination chemistry. Note that curli are a class of highly aggregated, extracellular fibers expressed by E. coli that are involved in biofilm formation. b) Large-scale and hierarchical assembly of diverse discreteor heterogeneous NOs on various interfaces such as 3D materials of irregular shapes. c) Patterned assembly of NOs on 2D substrates through programmable light regulation by coupling a light-sensing strain with predefined masks. d) Layer-by-layer assembly of NOs by temporally controlling the sequential addition of different NOs into the culture medium in coordination with dynamic biofilm growth. Figure 2. Large-scale and hierarchical assembly of red fluorescence QDs on various 2D surfaces of wide-ranging functionalities or 3D materials of complex shapes with the cell-enabled dynamic biofilm formation. a) Contact angle comparison betweena bare PTFE surface and a PTFE surface with biofilm coatings (left), digital camera images (middle), and corresponding fluorescence intensity comparison (right) of the QDs assembled on a PTFE surface with (bottom)/without (top) biofilm coatings. b) Assembly of CdSeS@ZnS QDs on various 2D flat surfaces, 3D material interfaces, and internal curved surfaces made of different materials. c) Assembly of CdSeS@ZnS QDs on flexible or foldable substrates, including PET substrates, cellulose paper, and cotton threads, respectively. d) The scalable assembly and immobilization of CdSeS@ZnS QDs on planar surfaces of polystyrene Petri dish or 3D interfaces of polyhedron empty ball (industrial filler) of different sizes. e) The hierarchy of assembled CdSeS@ZnS QDs structures across multiple length scales, revealed by fluorescence microscopy imaging, SEM, TEM, HRTEM, HAADF, and EDS mapping. The top left is a schematic of the hierarchy of assembled structures. Note that Teller rosette and pall ring is made of polypropylene (PP), and K1 filler is made of high-density polyethylene. Biofilm growth was carried out with E. coli TcReceiver/CsgAHis cells using Tc as inducer in M63 media, supplemented with red QDs. If not specifically noted, all the digital images were taken under UV light. Figure 3. The directed assembly of diverse types of inorganic NOs or complex heterogeneous structures through the a–i) designer cell-enabled dynamic biofilm formation and j–n) relevant applications:. a–c) single type of NOs assembled on biofilms using Au NPs as a representative); d–f) complex assembly of heterogeneous structures comprising two different types of NOs using Au NPs and red CdSeS@ZnS QDs as representatives; g–i) complexassembly of heterogeneous structures comprising three different types of NOs using Au NPs, CdS NRs, and red QDs as representatives. b, e, h) HAADF images. c, f, i) EDS mapping images, in which the red and blue colors refer to Cd and Au elements, respectively. j) Schematic showing preparation of conductive layers on biofilm-coated substrates through an electroless gold enhancement process. k) I–V curve comparison for Au NPs anchored biofilms on PET plate before and after gold layer enhancement. Dimension of the PET plate is 2×6 cm. l) Au layer enhanced biofilms on PET plate showing good conductivity, illustrated with a connected blue LED light. m, n) Demonstration of application of Interdigitated electrode arrays and a touch switch device: m) an interdigitated electrode arrays based on patterned Au layers on PET plate (inset) and its repeatable capacitance performance based on multiple times of finger touch; n) an LED touch switch device based on the capacitor shown in panel (m). Note that the bottom image in each of panels (a), (d), and (g) is the zoomed-in image shown in the white box of the top image. Scale bars: a, d, g) top, 1 μm and bottom, 50 nm; b, c) 150 nm; e, f) 10 nm; and h, i) 5 nm. Biofilm growth was carried out with E. coli TcReceiver/CsgAHis cells using Tc as inducer in M63 media, supplemented with various NOs. The structures assembled are dependent on the initial NO recipes supplemented to the culture media. Figure 4. Patterned assembly of fluorescent QDs through programed light regulation coupling E. coli lightReceiver/CsgAHis designer cells with predefined masks. a) Light-inducible genetic circuit based upon the pDawn-CmR-csgAHis plasmid construct. b) Histograms showing photoluminescence (PL) mapping comparison of QDs’ assembly at the bottom of a Petri dish with biofilms growing either under the blue light or dark condition after 20 and 36 h induction time. The inserted pictures showing the corresponding digital images of biofilm-immobilized QDs. Scale bars, 4 mm. c) The hierarchical features of patterned red QD structures assembled on planar plate across multiple length scales, revealed by fluorescence microscopy imaging, SEM and TEM. The top is a schematic showing the hierarchy of the assembled structures. d) Fluorescent images showing patterned assembly of QD structures. From left to right, the results were based on masks featured with patterned holes having diameters of 50, 100, and 200 μm, respectively. The final image is the result of green CdZnSeS@ZnS QDs based on a mask having the patterned holes with a diameter of 200 μm. d) Co-patterning of red and green QDs by simultaneous addition of red and green QDs into the culture media. e) Complexred QD patterns produced by coupling the light-inducible biofilm with a prestructured mask, featuring the morphology of a square, triangle, pentacle, and circle, respectively (from left to right). The inserted images showing the masks that were applied, with white dots representing holes created on the masks (each hole having a diameter of 0.45 mm). Scale bars, 5 mm. f) Red QDs’ pattern featuring a badge of ShanghaiTech University (bottom image). The upper image showing the mask applied for creating the red QDs’ patterning. Scale bars, 5mm. Note: Blue light promotes biofilm formation at the light exposure area, which displays red color as a result of red fluorescence features of the Co-NTA CdSeS@ZnS QDs co-assembled with dynamic biofilm formation. If not specifically noted, all the digital images were taken under UV light. Figure 5. Construction of single-layer or multilayered QDs thin film structures based on layer-by-layer assembly of NOs through temporally controlling the sequential addition of NOs into the culture in coordination with dynamic biofilm assembly. a) Fluorescence microscopy images of single-layer assembly of blue QDs (thickness, 19 μm). b) Double-layer sequential assembly of blue QDs (thickness, 19 μm) (bottom) and green QDs (thickness, 17 μm) (top). c) Triple-layer sequential assembly of blue QDs (thickness, 16 μm) (bottom), green QDs (thickness, 13 μm) (middle), and red QDs (thickness, 13 μm) (top). d) Double-layer sequential assembly of blue (thickness, 13 μm) (bottom), and mixed green and red QDs (thickness, 33 μm) (top). e, f) Structural comparison of double-layer sequential assembly of blue and red QDs on planar and curved surfaces: e) layers of blue QDs (thickness, 19μm) (bottom) and red QDs (thickness, 16 μm) (top) on planar surface; f) layers of blue QDs (thickness, 13 μm) (bottom) and red QDs (thickness, 12 μm) (top) oncurved surface. The top image in panels (e) and (f) is a schematic showing double-layer sequential assembly of blue and red QDs on planar and curved surface, respectively. The top images in each of panels (a)–(d) and middle images in panels (e) and (f) are side-view images of the bottom images. Scalebars, 100 μm. Red, green, and blue QDs refer to CdSeS@ZnS, CdZnSeS@ZnS, and CdZnS@ZnS, respectively. Biofilm growth was carried out with E. coli TcReceiver/CsgAHis cells using Tc as inducer in M63 media, supplemented with various NOs. 该论文中，物质学院2015级博士生王新宇为第一作者，2017级博士生濮嘉华和安柏霖为共同第一作者，钟超为通讯作者，上科大为第一完成单位。物质学院宁志军教授课题组2016级博士生尚跃群参与了该项目，并在量子点的合成方面提供了指导和帮助。2014级本科生刘奕和2015级本科生巴方对该研究的样品拍摄提供了帮助。这项成果的取得体现了上科大强调交叉学科建设的重要性，也反映了上科大鼓励本科生勇于实践、积极参与科研活动的理念。 本文文献资料均已上传至科学之理QQ群(QQ: 574543664)，入群获取资料请注明：科学之理。文件名：Programming Cells for Dynamic Assembly of Inorganic Nano-objects with Spatiotemporal Control.
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