Volume 51 Issue 11
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Zhong Wencheng, Guo Wenfeng, Shang Li. Advances in biological imaging applications of fluorescent gold nanoclusters (invited)[J]. Infrared and Laser Engineering, 2022, 51(11): 20220527. doi: 10.3788/IRLA20220527
Citation: Zhong Wencheng, Guo Wenfeng, Shang Li. Advances in biological imaging applications of fluorescent gold nanoclusters (invited)[J]. Infrared and Laser Engineering, 2022, 51(11): 20220527. doi: 10.3788/IRLA20220527

Advances in biological imaging applications of fluorescent gold nanoclusters (invited)

doi: 10.3788/IRLA20220527
  • Received Date: 2022-07-29
  • Rev Recd Date: 2022-09-30
  • Publish Date: 2022-11-30
  • In recent years, due to the unique advantages such as excellent fluorescence properties, ultra-small size, precise chemical structure and good biocompatibility, gold nanoclusters (AuNCs) have become a kind of emerging fluorescent nanoprobe of great concern. In order to promote the application of AuNCs in fluorescence imaging, researchers have been devoted to designing preparation strategies for various high-performance fluorescent AuNCs. Based on the continuous understanding of the structure and luminescence mechanism of AuNCs, strategies such as enhancing the fluorescence quantum yield and cellular uptake of AuNCs have been proposed and applied to enhance the cellular imaging ability of AuNCs. These strategies greatly improve their potential as fluorescent imaging probes. Furthermore, fluorescent AuNCs are also utilized in advanced fluorescence imaging technologies such as fluorescence lifetime imaging and multi-photon fluorescence imaging. In addition, AuNCs with near-infrared II fluorescence have greatly promoted their application for in vivo imaging recently. This article summarizes the preparation methods of fluorescent AuNCs probes, reviews strategies to improve the fluorescence cellular imaging ability of AuNCs, and introduces the latest progress in the application of AuNCs in fluorescence imaging and also the challenges and future developments in the field.
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Advances in biological imaging applications of fluorescent gold nanoclusters (invited)

doi: 10.3788/IRLA20220527
  • State Key Laboratory of Solidification Processing, School of Materials Science and Engineering, Northwestern Polytechnical University, Xi’an 710072, China

Abstract: In recent years, due to the unique advantages such as excellent fluorescence properties, ultra-small size, precise chemical structure and good biocompatibility, gold nanoclusters (AuNCs) have become a kind of emerging fluorescent nanoprobe of great concern. In order to promote the application of AuNCs in fluorescence imaging, researchers have been devoted to designing preparation strategies for various high-performance fluorescent AuNCs. Based on the continuous understanding of the structure and luminescence mechanism of AuNCs, strategies such as enhancing the fluorescence quantum yield and cellular uptake of AuNCs have been proposed and applied to enhance the cellular imaging ability of AuNCs. These strategies greatly improve their potential as fluorescent imaging probes. Furthermore, fluorescent AuNCs are also utilized in advanced fluorescence imaging technologies such as fluorescence lifetime imaging and multi-photon fluorescence imaging. In addition, AuNCs with near-infrared II fluorescence have greatly promoted their application for in vivo imaging recently. This article summarizes the preparation methods of fluorescent AuNCs probes, reviews strategies to improve the fluorescence cellular imaging ability of AuNCs, and introduces the latest progress in the application of AuNCs in fluorescence imaging and also the challenges and future developments in the field.

    • AuNCs一般由数个到数百个金原子构成,具有超小的尺寸,是一类近些年受到广泛关注的新型纳米材料[1-5]。因其超小尺寸、优异的荧光性质、良好的生物相容性与光稳定性等特点,AuNCs在传感[6]、成像[7]、疾病诊疗[8]与催化[9]等领域中均展现了极大的应用前景。目前常用的荧光AuNCs的制备方法为“自下而上”法,即在配体保护下将三价的Au离子还原形成AuNCs,且以其中的配体辅助还原法最为常见[10-12]

      配体辅助法就是指在配体存在的条件下,使用还原剂将金离子还原形成配体包覆的AuNCs的过程[13-14]。如图1所示,该方法中最重要的配体可以有多种选择,包括聚合物[15]、DNA[16]、硫醇分子[17] 以及蛋白质[18]等。配体辅助法具有以下优点:一方面,选取水溶性良好的配体用于合成AuNCs可以显著改善AuNCs的水溶性、稳定性;另一方面,可以通过对金核表面的配体进行修饰以改变AuNCs的理化性质。近年来, AuNCs在细胞、组织、活体层面荧光成像的应用获得了极大的进步。然而,面对更为复杂的荧光成像需求与新兴荧光成像技术,如何进一步地提高荧光成像效果,进而拓展AuNCs在新兴荧光成像技术领域的应用仍是一个值得关注的课题。

      Figure 1.  Synthesis of AuNCs by using (a) polymers[15], (b) DNA[16], (c) thiol molecules[17] and (d) proteins[18] as templates

    • 在细胞成像应用中,理想的荧光探针应具备高量子产率、高信噪比、高特异性等特点[19-20]。为了满足这些要求以实现较好的成像效果,如何精确设计和制备高性能荧光AuNCs探针至为关键。虽然在文献中已有大量报道将AuNCs作为新兴成像探针应用于荧光成像,然而其进一步的应用拓展仍受到荧光波长短、荧光量子产率低及亚细胞器靶向性差等因素的限制。为了解决这些问题,研究者们提出了多种有效的AuNCs功能化设计与制备策略,并取得了较为显著的进展。

    • 当AuNCs的荧光量子产率显著高于细胞自荧光物质时,AuNCs成像的信噪比将得到明显的提高,进而增强其细胞内荧光成像性能。为了有效地提高AuNCs的荧光量子产率,需要对该类材料的发光机理及其构效关系有一个清晰的认识。基于AuNCs精确的分子结构,研究者通过系统调控AuNCs的原子组成和配体性质等,深入研究了多种因素对于AuNCs荧光的影响,初步阐明了AuNCs的发光机理[21-22]。目前,关于AuNCs的发光机理的解释中被广泛认可的观点是,AuNCs的荧光来源于小尺寸Au核引起的离散电子能级,以及Au核-表面配体的电子传导等的综合效应。通过调控Au核的电子能级、增强Au核与配体之间的电子传导或者限制表面配体的自由度,以及利用AuNCs的聚集诱导发光性质等途径可以有效地提高AuNCs的荧光量子产率。因此,根据目前对AuNCs光致发光机理的理解,研究者们提出了多种可有效提高AuNCs荧光量子产率的设计与制备策略,如图2所示。

      当前,普遍采用的提升AuNCs的荧光量子产率的方式主要可以分为合金掺杂、表面配体调控、自组装以及聚集诱导发光等。在合金掺杂策略中最常用的合金元素为银,这主要是由于Au与Ag性质相近(同属IB族元素)且晶格适配。2014年,汪恕欣等人使用Ag掺杂策略将AuNCs的荧光量子产率提高到原先的200倍(图2 (a)[23]。除了Ag掺杂之外,姚传好等人采用Cd掺杂策略改变了金核的表面原子结构,从而显著提升了AuNCs的荧光量子产率[24]。由于AuNCs的性质与金核及表面配体密切相关,除了对AuNCs的金核进行调控外,AuNCs荧光量子产率的提升也可以通过配体改性实现。Chen Zhan课题组以谷胱甘肽(GSH)和组氨酸为共配体,得到了比单独组氨酸为配体所合成AuNCs荧光量子产率更高的产物[25]。除此之外,通过限制AuNCs表面配体的自由度也可以显著提升AuNCs的荧光量子产率。例如,陈伟课题组以6-氮杂-2-硫代胸腺嘧啶(ATT)作为配体合成AuNCs,通过加入精氨酸与配体之间形成氢键,实现对配体灵活度的限制和簇内电子传导的增强,从而制备得到了超高荧光量子产率(65%)的绿色荧光AuNCs (图2(b)[26],并在后续工作中基于相同原理以双席夫碱类分子增强AuNCs荧光,得到了量子产率为48%的红色荧光AuNCs[27]

      谢建平课题组于2012年报道了AuNCs的溶剂致聚集诱导发光现象,成功制备了荧光量子产率为15%的AuNCs聚集体(图2 (c)[28]。然而,在细胞成像的过程中,溶剂致聚集诱导发光现象在细胞环境、溶剂稀释等因素的影响下易失效,因此需要稳定的AuNCs聚集体制备方法。目前广泛采用的制备聚集体的方法为自组装策略,即将AuNCs组装为纳米片、纳米凝胶以及合成纳米颗粒等不同形貌。在各种组装策略中,基于各组分的正负电荷之间的静电作用所构建的AuNCs自组装体是较常采用的方法。例如,2016年,Xavier Le Guével课题组和谢建平课题组分别报道了以正电聚合物聚烯丙基胺盐酸盐(PAH)与壳聚糖诱导AuNCs自组装,结果表明得到的自组装纳米颗粒均具有更高的荧光量子产率(图2 (d)[29-30]。除此之外,基于主客体相互作用的超分子自组装也可以用于提高AuNCs的荧光量子产率[31-32]

      Figure 2.  Methods to improve the quantum yield of AuNCs: (a) Alloy doping in Au core[23]; (b) Surface ligand engineering[26]; (c), (d) Aggregation-induced emission and AuNCs-based self-assemblies[28-29]

    • 增强AuNCs荧光成像过程中的信噪比的另一种方法是提高细胞对AuNCs的摄取效率。为了实现高的细胞摄取效率,阐明AuNCs的细胞内化机制具有较为重要的意义。2014年,Nienhaus课题组通过荧光成像详细研究了HeLa细胞对AuNCs的内化过程(图3(a)[33],发现在该过程中具有超小尺寸的AuNCs会先富集在细胞膜表面,进而主要通过网格蛋白介导的内吞机制和大胞饮作用进入细胞。作者还进一步通过荧光寿命成像技术原位监控了AuNCs的胞内稳定性[34],为设计开发高稳定、高成像性能的AuNCs提供了重要的基础。

      除此之外,提高AuNCs的细胞摄取率还可以通过自组装[35]、细胞靶向配体修饰[36]以及表面正电荷修饰等方法[37]。由于细胞膜的表面呈负电位,因此,相比表面带负电的AuNCs,表面正电荷修饰的AuNCs更易于与细胞膜结合,从而提高AuNCs的细胞摄取率。然而,自组装与表面正电荷修饰虽然可以提高大部分细胞对于AuNCs的摄取率,但是无法区分不同的细胞类别,对于基于荧光成像的精准诊疗应用缺乏指导意义。由于部分癌细胞表面具有比正常细胞更高的叶酸、糖受体的表达水平,因此通过修饰叶酸、糖类等特定生物分子不仅可以提高AuNCs在叶酸、糖类等受体过表达的癌细胞的摄取率,增强其荧光成像性能,也可以实现对此类癌细胞的特异性区分(图3(b)[38-39]。如图3 (c)所示,叶酸修饰的AuNCs在叶酸受体过表达的HeLa、KB细胞的摄取量明显高于未过表达的MG63、A549等细胞。

      Figure 3.  (a) Investigating the endocytosis of AuNCs by fluorescence imaging[33]; (b) Scheme of modification of folic acid on the surface of AuNCs[38]; (c) Differences in cellular uptake of unmodified AuNCs (SG) and folic acid-modified AuNCs (FA) in cells with different receptor expression levels by flow cytometry[38]

    • 随着AuNCs在细胞荧光成像方面的应用的逐步扩展,如何在亚细胞水平上控制AuNCs在细胞内的定位,进而实现精准的细胞器成像成为了研究者们普遍关注的问题。

      借鉴其他荧光探针的靶向细胞器策略,多种特异性靶向细胞器的分子(如三苯基膦等)被用于修饰Au-NCs表面以获得靶向细胞器的能力。例如,庞代文课题组通过特异性靶向细胞核的TAT多肽修饰AuNCs,从而赋予AuNCs靶向细胞核的能力[40]。朱满洲课题组在合成中加入带巯基的三苯基膦配体,将原先靶向溶酶体的AuNCs改造为可特异性靶向线粒体的AuNCs[41]。Yangang Wang课题组将靶向内质网的磺酰脲化合物修饰于AuNCs表面后,表现出较为明显的靶向内质网的能力[42]。这些具备特异性靶向性能的AuNCs为研究人员提供了进一步胞内精准成像的重要前提,借助于AuNCs的低毒性、优异的光稳定性等特点,使得长时间动态追踪特定细胞器的生物行为成为了可能。2021年,Yuan Wang等报道了一种可靶向线粒体的荧光AuNCs,且其荧光寿命与温度密切相关,从而利用荧光寿命技术揭示了细胞内不同区域线粒体的温度存在明显差异(图4[43]

      Figure 4.  AuNCs as fluorescence probes for (a) cellular imaging of mitochondria and (b) investigating temperature of mitochondria in different regions within a cell through fluorescence lifetime imaging[43]

    • 与传统的共聚焦荧光成像方式相比,荧光寿命成像、双光子荧光成像、超分辨成像等新的成像技术可以提供更高的信噪比和成像分辨率。目前,荧光AuNCs在双光子成像技术与荧光寿命成像的应用报道相对较多。相比生物体系的自荧光,AuNCs普遍具有较长的荧光寿命,因而可以很容易通过荧光寿命成像来有效区分探针的荧光和自荧光,从而实现荧光成像性能的提升。例如,尚利等人合成了近红外荧光AuNCs,在荧光寿命成像的过程中有效区分了强度信号无法区分的组分,并实现了基于荧光寿命的细胞内温度的实时监测(图5(a)[44-45]。双光子成像技术相比单光子成像技术而言具有更低的光子能量,且不易激发细胞内的自荧光组分。因此,具有优异双光子吸收性能的AuNCs在双光子甚至多光子成像技术下展现出了更为出色的细胞荧光成像能力(图5(b)[46-47]

      Figure 5.  Fluorescent AuNCs for (a) fluorescence lifetime imaging[44], (b) multi-photon fluorescence imaging[47], (c) STED imaging[48] and (d) super-resolution radial fluctuations imaging[49]

      相比而言,目前关于AuNCs在超分辨成像技术领域的应用报道较少。其中,Hongwei Yang等与Aditya Yadav等分别报道了ATT/Arg-AuNCs(图5(c))与BSA-AuNCs(图5(d))在受激辐射损耗(STED)成像技术与超分辨径向涨落成像方面的应用,分辨率分别达到70 nm与59 nm,显著提升了AuNCs在细胞内荧光成像的分辨率[48-49]。由于超分辨成像技术在生物成像领域的巨大应用前景,发展能更好满足超分辨荧光探针需求的AuNCs将会是一个十分值得研究的方向。

    • 早期研究中制备的AuNCs的荧光大多在可见光波段,且荧光量子产率较低,在进行活体荧光成像时,来自细胞、组织自荧光的干扰将会显著地影响AuNCs成像的效果,在一定程度上限制了AuNCs在活体水平的成像应用。为了避开几乎覆盖整个可见光波段的生物自荧光(400~700 nm),开发具有近红外一区荧光(650~900 nm)的AuNCs成为了较为广泛采用的一个解决方案。目前,研究人员已经设计合成了多种具有近红外一区荧光的AuNCs,并将其成功用于生物荧光成像[50]。然而,具有近红外一区荧光的AuNCs在活体水平成像层面仍存在组织穿透性差、信噪比较低等问题。这是由于与细胞成像应用相比,活体水平的荧光成像更具挑战。相较细胞而言,生物组织具有更强的自荧光,而且可见光的组织穿透深度有限。近年来,研究人员发现部分AuNCs的荧光波段可以延伸至近红外二区(NIR-II,发射波长为1 000~1 700 nm),显著推动了荧光AuNCs在活体成像领域方面的应用潜能[51-58]。目前,已成功用于合成具有NIR-II荧光性质的AuNCs 的配体包括硫醇小分子、多肽、蛋白质以及超分子等。如图6所示,具有NIR-II荧光发射的AuNCs已被成功应用于活体内各种器官的成像,其中包括胃肠道[53]、骨[54]、肾[55]、血管[56]等。

      Figure 6.  AuNCs with NIR-II emission for fluorescence imaging of (a) gastrointestinal[53], (b) bone[54], (c) renal metabolism[55] and (d) blood vessel in vivo[56]

      在借助NIR-II成像技术实现了更高穿透深度、更低背景噪音的前提下,研究人员进一步在AuNCs的成像应用方面实现了新的突破。例如,Xavier Le Guével等通过调控AuNCs的NIR-II发光性质,实现了活体水平的三维NIR-II荧光成像[51]。喻志强等将NIR-II荧光的AuNCs与顺铂药物结合,成功用于同步的荧光成像和肿瘤治疗[52]。宋晓荣等将发射波长为1050 nm 的AuNCs组装于Ln纳米颗粒(发射波长为1550 nm)表面,实现了活体内比率荧光传感硫化氢(图7(a)[57]。杜国庆等通过将Gd修饰到AuNCs的表面,成功实现了对肿瘤的NIR-II荧光成像与核磁共振成像的双模态成像,进而用于指导肿瘤的放射治疗(图7(b)[58]。虽然具有NIR-II荧光发射的AuNCs在活体成像与应用方面已经取得了显著的成果,然而目前对于这些AuNCs的体内精准靶向仍存在一定的挑战[59-60]。如何进一步地控制NIR-II荧光的AuNCs在体内的代谢途径与稳定性,无疑将有助于未来在活体成像方面更深层次的应用。

      Figure 7.  (a) In vivo ratiometric fluorescence sensing of hydrogen sulfide by using NIR-II fluorescent AuNCs combined with Ln nanoparticles[57]; (b) NIR-II fluorescent AuNCs doped with Gd for MRI and NIR-II dual mode imaging[58]

    • 相比其他荧光成像探针而言,AuNCs的组成精确可调、光稳定性好与生物相容性优异等优势使得其在荧光成像领域具有较好的应用前景。研究者通过提高荧光量子产率、细胞摄取率等多种针对性的设计、制备策略,显著地推动了AuNCs在细胞、活体等层面成像的应用。其中,开发了具有NIR-II荧光的Au-NCs,使得其在活体荧光成像的应用潜能得到进一步的提升。然而,目前AuNCs在荧光成像方面的应用仍然存在诸多挑战:(1)从细胞层面上如何进一步提高AuNCs在荧光成像方面的精度、特异性,以及拓展AuNCs在多种荧光成像技术尤其超分辨成像方面的潜力;(2)在活体水平上如何精准控制AuNCs的器官靶向性及滞留时间,并在此基础上实现纳米探针成像、诊断、治疗的一体化;(3) AuNCs的发光机制对于指导AuNCs探针的精确设计及提高其荧光成像应用性能至关重要,目前对于AuNCs的荧光机制尚不明晰,仍需进一步在原子水平上阐明其结构和与荧光性能的内在关联。随着这些研究工作的不断深入,荧光AuNCs探针在生物成像及其他相关领域的应用势必会得到进一步的推动和拓展。

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