光学仪器  2018, Vol. 40 Issue (1): 13-18   PDF    
牛血清蛋白-Au配合物及其复合物制备及表征
王圣琼1,3,4, 孙畅2, 陶春先1,3,4, 韩朝霞1,3,4, 洪瑞金1,3,4, 林辉1,3,4, 张大伟1,3,4     
1. 上海理工大学 光电信息与计算机工程学院, 上海 200093;
2. 复旦大学 高分子科学系, 上海 200433;
3. 上海理工大学 教育部光学仪器与系统工程研究中心, 上海 200093;
4. 上海理工大学 上海市现代光学系统重点实验室, 上海 200093
摘要: 以牛血清蛋白(bovine serum albumin,BSA)、氯金酸(HAuCl4)、NaOH等为原料,采用一锅合成法制备了牛血清蛋白-Au配合物(BSA-Au),观察到了样品的宽带红光荧光发射。通过库仑力作用将BSA-Au与表面带有正电荷的金纳米颗粒进行复合,对纳米复合物的吸收光谱及荧光光谱特性进行了研究。实验结果表明,在本实验条件下,金纳米颗粒虽然增强了复合物样品在可见光区域的光吸收,但对BSA-Au红光发光有猝灭作用。
关键词: 牛血清蛋白     亚纳米尺度金簇     荧光光谱    
Preparation and characterization of bovine serum albumin(BSA)-Au complexes and the Au nanoparticle@BSA-Au nanocomposites
WANG Shengqiong1,3,4, SUN Chang2, TAO Chunxian1,3,4, HAN Zhaoxia1,3,4, HONNG Ruijin1,3,4, LIN Hui1,3,4, ZHANG Dawei1,3,4     
1. School of Optical-Electrical and Computer Engineering, University of Shanghai for Science and Technology, Shanghai 200093, China;
2. Department of Macromolecular Science, Fudan University, Shanghai 200433, China;
3. Engineering Research Center of Optical Instruments and Systems(MOE), University of Shanghai for Science and Technology, Shanghai 200093, China;
4. Shanghai Key Laboratory of Modern Optical System, University of Shanghai for Science and Technology, Shanghai 200093, China
Abstract: Bovine serum albumin(BSA)-Au complexes were synthesized by the one-pot method with BSA, HAuCl4 and NaOH as the starting materials.Red photoluminescence was observed from the sample.Au nanoparticle@BSA-Au composites were fabricated via the Coulomb interaction between the negatively charged BSA-Au and the positively charged Au nanoparticles.Optical absorption and photoluminesence properties of the samples were investigated.The experimental results show that optical absorption in the visible region was enhanced for the Au nanoparticle@BSA-Au composites, due to the localized surface plasmon resonance.However, the luminescence of BSA-Au was quenched.Further work on improving the structure of the Au nanoparticle@BSA-Au composites needs to be conducted.
Key words: bovine serum albumin     sub-nm gold cluster     photoluminescence spectra    
引言

以金、银为代表的亚纳米尺度贵金属发光结构由于其良好的物理化学稳定性、较高的荧光量子产额、在一定条件下的开/关可控发光等特性, 使得其在生物荧光探针[1]、化学传感器[2-3]、光存储/光编码[4-5]、无稀土荧光粉[6]等领域显示出了很好的应用前景。由于亚纳米尺度金、银的尺寸非常小(一般<2 nm), 通常需要基质材料加以稳定以防止其团聚, 常见的基质材料有硫醇[7-9]、半胱氨酸[10-11]、DNA[12]、蛋白质[13]、聚合物[14-16]等, 对于无机基质材料, 目前有见报道的只有沸石[17-22]和玻璃[23-24]两种。

在众多有机配体材料中, 牛血清蛋白由于具有优异的生物兼容性、水溶性、低廉的价格, 以及合成获得的牛血清蛋白-Au配合物(BSA-Au)样品具有较高的发光强度而被广泛研究[25]

以往, 多数研究者认为BSA-Au样品的发光机理是基于金属自由电子理论和久堡模型, 即当金的尺寸减小到与其费米波长相当时, 其原本准连续的电子能级会发生劈裂, 当最高占据分子轨道(highest occupied molecular orbital, HOMO)与最低未占据分子轨道(lowest unoccupied molecular orbital, LUMO)之间的能隙劈裂到可以产生辐射发光电子跃迁时, 对应金的临界尺寸约为2 nm[26]。最近, 我们的实验结果表明, BSA-Au样品的宽带红光发射很可能不是起源于量子尺寸限制效应, 而很可能是来自由配体到Au(Ⅰ)离子的电荷转移发光[27]。电荷转移发光通常具有较大的斯托克斯位移, 对于BSA-Au样品, 其最强的激发峰位于紫外区域, 而紫外光对细胞有杀伤力, 对人体组织有一定危害。因此, 增强BSA-Au样品在长波区域的激发效率有益于实际应用。基于局域表面等离激元效应, 通过调节金纳米颗粒的尺寸、形状等参数, 可以调控其在可见光到近红外波段的吸收性能。而将具有适当吸收的金纳米颗粒与BSA-Au样品进行复合, 预期能够增强BSA-Au样品在长波区域的光吸收, 同时局域表面等离激元共振效应也可能对BSA-Au的荧光强度有所增强。

本文采用一锅合成法制备了BSA-Au样品, 将其与进行过表面修饰而带有正电荷的Au纳米颗粒通过库仑力作用形成复合物, 测试和分析了复合物样品的吸收和荧光光谱, 并对荧光光谱变化进行了分析讨论。

1 原理

由于BSA分子中存在的巯基与重金属离子(如Au(Ⅰ)离子)间存在很强的相互作用, 因此可以合成出BSA-Au配合物。而对于BSA-Au配合物, 因其在可见光区域的吸收相比其紫外光区域的吸收较弱, 本文通过库仑力作用将带负电的BSA-Au与表面带有正电荷的金纳米颗粒进行复合, 并通过金纳米颗粒的局域表面等离激元共振来增强BSA-Au配合物在可见光区域的吸收。

2 样品制备及测试

所有化学试剂均采购自日本和光化学试剂有限公司, 主要采用氯金酸(HAuCl4·H2O)、牛血清蛋白(BSA)、氢氧化钠(NaOH)等试剂合成BSA-Au样品。

2.1 样品制备

(1) BSA-Au(Ⅰ)样品的制备

采用文献[25]中报道一锅法制备BSA-Au样品。合成后的样品溶液采用8~14 kDa((1 Da=1 u=(1.660 540 2±0.000 001 0)×10-27 kg)的半透膜对去离子水进行渗析, 每隔4 h更换1次去离子水(DI水), 以去除在溶液中游离的Na+离子、OH-离子、Au(Ⅰ)离子、Au(Ⅲ)离子和蛋白质小碎片等杂质[28], 直至BSA-Au(Ⅰ)溶液的pH到达7时停止渗析。

(2) Au纳米颗粒@BSA-Au(Ⅰ)复合物样品的制备

将500 μL和2.5 mL表面带有正电荷的金纳米颗粒(约1×1016个/mL)溶液分别缓慢滴入2份搅拌中的500 μL的BSA-Au水溶液中, 这样带有负电荷的BSA-Au样品将会与表面带有正电荷的金纳米颗粒通过库仑力作用结合到一起, 得到复合物样品, 记为Au NP@BSA-Au, 分别简记为C1和C2。

2.2 测试表征

采用基质辅助激光解析电离-飞行时间质谱仪(MALDI-TOF MS)测试了BSA-Au样品的金含量。采用紫外-可见分光光度计测试了样品的吸收光谱, 测试范围为300~800 nm。采用Horiba Fluorolog 3型荧光光谱仪测试了样品的荧光光谱。

3 结果与讨论

图 1是纯BSA与BSA-Au样品的MALDI-TOF MS图谱, 从图中可以看出:纯BSA对应的质荷比约为66 500;而对于BSA-Au样品, 样品的信号峰出现了宽化, 且其峰值对应的质荷比减小至约65 900, 这是由于BSA-Au合成过程中经历了强酸和强碱环境, 导致BSA分子以小碎片形式损失掉一部分质量所造成的。由此可见, 通过MALDI-TOF MS精确分析BSA-Au样品中金的精确含量存在一定的困难。

图 1 纯BSA与BSA-Au样品的MALDI-TOF MS图谱 Figure 1 MALDI-TOF MS measurement for the pure BSA and BSA-Au sample

图 2是BSA-Au样品以及Au NP@BSA-Au样品(C1和C2)的吸收光谱。图 2(a)中BSA-Au样品在可见光波段并无显著吸收峰, 随着波长的减小, 吸收逐渐增强; 对于Au NP@BSA-Au样品, 位于536 nm和538 nm的吸收峰对应于金纳米颗粒的局域表面等离激元共振吸收。而在图 2(b)的荧光光谱中, Au NP@BSA-Au复合物的荧光强度相比BSA-Au(Ⅰ)样品大幅减弱, 产生了严重的荧光猝灭现象。

图 2 BSA-Au, Au NP@BSA-Au样品的吸收光谱和荧光光谱 Figure 2 Optical absorption and photoluminescence spectra of the BSA-Au and Au NP@BSA-Au samples

导致BSA-Au样品产生荧光猝灭的原因可能是:在BSA-Au与金纳米颗粒结合过程中, 正负电荷抵消掉一部分, 而前期的研究结果表明, 样品所携带负电荷的数量与BSA-Au样品红光荧光强度正相关[27], 因此导致了荧光猝灭。

图 3为BSA-Au样品以及Au NP@BSA-Au样品(C1和C2)的激发-发射3D等高图。可以看出, Au NP@BSA-Au样品的最强激发峰仍位于紫外区域(约375 nm处), 即虽然金纳米颗粒通过表面等离激元共振增强了Au NP@BSA-Au样品在可见光区域的光吸收, 但并没有有效增强BSA-Au在可见光区域的激发效率。

图 3 不同样品的激发-发射3D等高图 Figure 3 Excitation-emission 3D contours of different samples
4 结论

制备了BSA-Au和BSA-Au/金纳米颗粒复合物样品, 采用MALDI-TOF MS分析了BSA-Au样品中的金含量, 并分析了研究中存在的困难。测试了样品的吸收光谱和荧光光谱。实验结果表明, 在本文工作实验条件下, 金纳米颗粒虽然增强了复合物样品在可见光区域的光吸收, 但对BSA-Au红光发光有猝灭作用, 后续工作需要改进复合物的结构设计。

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