基于表面增强拉曼光谱(SERS)拟建立一种适用于水产鱼制品中组胺含量的快速检测方法。 采用银纳米颗粒(Ag NPs)作为活性基底和氯化钠溶液作为聚集剂获取组胺的SERS特征峰, 并结合线性回归算法对鱼肉中的组胺含量进行测定分析。 首先对固体组胺、 组胺水溶液以及鱼肉提取液中组胺的SERS特征峰及归属进行表征分析, 然后对以Ag NPs的浓缩倍数与氯化钠溶液的浓度作为SERS基底的反应条件进行优化, 最后在该优化条件下对鱼肉中组胺进行定量分析。 结果表明, Ag NPs在400 nm处有最大吸光度, 通过透射电子显微镜观察颗粒的形状主要为球形, 均匀尺寸为30 nm左右。 利用4-巯基苯甲酸作为探针分子对其进行拉曼测试, 所得拉曼峰具有良好的重复性, 且拉曼强度很高。 因此该活性基底的合成方法不仅用时少、 易操作, 且合成的Ag NPs可作为可靠的增强基底应用于SERS试验中。 此外通过紫外-可见分光光度计检测得出氯化钠溶液使Ag NPs在溶液中发生团聚, 形成热点, 可实现SERS信号增强。 固体组胺的拉曼光谱图反映出1 167 cm-1处出现的特征峰主要是由N—H面内弯曲引起的; 1 236 cm-1处的特征峰主要是咪唑中C—H平面内弯曲和环呼吸引起的; 1 291 cm-1处主要与环伸展有关; 1 474 cm-1处的特征峰主要是由咪唑N—H面内弯曲振动和环伸展引起的。 优化反应条件在Ag NPs的浓缩倍数为15倍、 氯化钠溶液浓度为1 mol·L-1时表现出最高的增强效应, 并在该优化条件下检测了浓度为5～250 mg·L-1的组胺水溶液, 得出在该优化条件下检测到组胺水溶液的最低浓度为5 mg·L-1。 同时在该优化条件下采集了10～100 mg·L-1范围的鱼肉提取液中组胺的SERS光谱, 并建立组胺溶液的特征拉曼位移峰强度与浓度之间的线性回归模型。 得出在1 180, 1 258和1 425 cm-1处的特征峰与对应的拉曼峰强度值所建立的标准曲线有良好的线性关系(R2=0.918 1～0.947 3), 通过比较得出在1 258 cm-1处特征拉曼位移峰强度的R2值最大, 且在鱼肉中组胺的最低检测浓度为10 mg·L-1, 远低于国标中水产品中组胺最大限量检测限50 mg·L-1。 因此选择1 258 cm-1处的标准曲线进行进一步的组胺检测。 最后通过对鱼肉提取液中添加组胺对该标准曲线进行检测验证, 得到回收率在100%～111%之间。 且通过高效液相色谱法验证该方法具有适用性。 由此表明选取银纳米颗粒作为活性基底、 氯化钠溶液作为聚集剂的表面增强拉曼光谱技术结合线性回归法建立标准曲线用于快速检测鱼肉中的组胺是可行且准确, 这为在鱼肉中的组胺含量的快速定量分析提供了参考依据。

A rapid method for the detection of histamine in fish products was developed in this study based on Surface-enhanced Raman spectroscopy (SERS). Silver nanoparticles (Ag NPs) were used as the active substrate and sodium chloride solution as an aggregating agent to obtain histamine SERS peaks. The histamine content in fish extracts was determined by linear regression algorithm. In this research, the normal Raman spectra of histamine powder, the SERS characteristic peaks and vibrational assignment of histamine standard solutions as well as histamine in fish extracts were analyzed. The SERS reaction conditions for the concentration of silver nanoparticles and sodium chloride solution were optimized. Lastly, the optimization conditions were used to quantitatively analyze the histamine in fish extracts. The results showed that the silver nanoparticles had the maximum absorbance at 400 nm. The shape of the nanoparticles was mainly spherical and the average size was about 30 nm. Then using 4-MBA as a probe molecule to detect the stability and sensitivity of Ag NPs and obtained Raman peak has good repeatability and high Raman intensity. Therefore, not only is the synthesis method of the active substrate less time-consuming and easy to operate, but the synthesized Ag NPs can be used as a reliable reinforcing substrate in the SERS test. In addition, it was proved by UV-Vis spectrophotometer that the NaCl solution can arouse the agglomeration of Ag NPs to form hot spots, which can enhance SERS signal. The Raman spectrum of solid histamine reflects that the characteristic peak appeared at 1 167 cm-1 due to the in-plane bending of N-H, the in-plane bending of imidazole in C—H plane and ring breathing (1 236 cm-1), the ring stretching (1 291 cm-1), the in-plane bending vibration of imidazole N—H and ring stretching (1 474 cm-1). Then the optimized reaction conditions showed the highest enhancement effect when the concentration multiple of silver nanoparticle was 15× and the concentration of sodium chloride solution was 1 mol·L-1, and the optimal condition was detected at the of 5～250 mg·L-1 histamine aqueous solution, of which the lowest concentration was detected at 5 mg·L-1. At the same time, the SERS spectra of histamine in fish extracts ranging from 10 to 100 mg·L-1 were detected under the optimized conditions. It was revealed that 3 calibration curves established at the characteristic peaks at 1 180, 1 258, 1 425 cm-1 corresponding Raman peak intensities had a good linear relationship when the concentration of histamine in fish extracts ranged from 10 to 100 mg·L-1 with R2= 0.918 1～0.947 3, of which the R2 value of the prominent peak at 1 258 cm-1 was the largest. The lowest concentration detected in fish was 10 mg·L-1, much lower than maximum limit of detection of histamine in the national standard. The calibration curve had superior accuracy with the recovery between 100%～111%. And the applicability of the method was verified by high performance liquid chromatography (HPLC). The experimental results showed that surface-enhanced Raman spectroscopy using silver nanoparticles as an active substrate and sodium chloride solution as an aggregator coupled with linear regression to establish a standard curve for rapid detection of histamine in fish is feasible and accurate, which provides a reference basis for rapid quantitative analysis of histamine content in fish products.