Abstract
Abstract in English, Chinese Biogenic amines (BAs) represent a class of potentially harmful substances in foods and medicines. Their content is thus an important indicator of proper hygiene in food preparation, and purity of medicines. It is of great practical significance to establish accurate and sensitive detection of BAs in food and drugs. In this study, a high performance liquid chromatography (HPLC) method was developed for the simultaneous detection of multiple BAs in fish, pork and antibiotics based on aptamer signal replacement and cyclic amplification strategy. First, non-fluorescent targets were converted into fluorescent nucleic acid probes using a two-step replacement process. Subsequently, a large number of nucleic acid probes with different lengths and base sequences were generated using a double-stranded specific nuclease-assisted signal amplification strategy. Finally, various BAs in real samples were accurately identified using an HPLC platform. The influence of base sequence and nucleic acid probe length on separation via HPLC was studied to improve discrimination among fluorescent signals. Four different sequences were selected as tails to the DNA probe, and their retention times increased in turn. Experimental conditions, including column temperature, flow rate, gradient elution process, reaction temperature, and incubation time, were optimized by orthogonal experiments to further improve signal separation efficiency. Specifically, the methanol gradient was changed from 10% to 20% during 0-20 min, 35 ℃ of column temperature and 1.0 mL/min of flow rate were chosen as the HPLC conditions. The final resolution of chromatographic peaks was 3.44, 3.59 and 2.37, indicating complete separation between peaks. Optimal incubation time for BA capture by aptamer was 120 min, and optimal dosage of duplex specific nuclease (DSN) and Mg2+were 0.9 U and 30 mmol/L. The optimal pH, incubation temperature, and DSN incubation time were 7.0, 40 ℃ and 210 min, respectively. The proposed method exhibited high sensitivity towards BAs, with a linear range of 1 pmol/L-1 μmol/L, and the limits of detection of tyramine, histamine, spermine, and tryptamine were 0.25, 0.21, 0.27 and 0.19 pmol/L, respectively. The feasibility of this method was verified, and contrast experiments indicated that it could achieve highly selective detection of four BAs in one run. The applicability of this integrated method was also investigated for the detection of real samples (gentamycin sulfate, fish and pork). To assess the matrix effect, each BA with different concentrations were spiked into real fish and pork samples. Relative recoveries and relative standard deviations (RSDs) ranged from 101.2% to 104.5% and from 1.5% to 4.3%, respectively. The above detection results for real samples showed that this method could accurately capture, separate, and identify BAs in complex matrix samples. This strategy can effectively improve analyte selectivity and reduce the matrix effect. This assay is thus expected to provide a new approach for food and drug analyses. 生物胺的含量是衡量食品卫生状况和药物纯度的重要标志之一,建立食品药品中生物胺的精准、灵敏检测具有重要的实际意义。该文基于核酸适配体置换生物胺信号源并结合荧光信号循环扩增的策略,建立了一种新型的同时检测鱼肉、猪肉和抗生素中4种生物胺的高效液相色谱法(HPLC)。首先通过两步信号置换,将无荧光信号的目标物转换为有荧光信号的核酸探针;再结合双链特异性核酸酶辅助信号扩增策略,获取大量不同长度和碱基序列的核酸探针;最后借助HPLC平台实现实际样品中多种生物胺信号的精确识别。文章研究了核酸探针的碱基序列和长度对出峰时间和前后顺序的影响,以提高荧光信号的区分度。通过正交实验探讨了柱温、流速和梯度洗脱过程、反应温度、孵化时间等对信号分离的影响,确定最优条件,提高信号的分离效率。该方法对目标物酪胺、组胺、精胺和色胺的检出限分别为0.25、0.21、0.27和0.19 pmol/L,线性范围为1 pmol/L~1 μmol/L。通过对硫酸大庆霉素、鱼肉和猪肉样品中生物胺含量进行检测,研究了该方法检测实际样品的可行性。该方法可精准识别、捕获和分离复杂基质样品中的生物胺组分,能有效提高对目标分析物的选择性,并降低实际样品中的基质干扰,有望为食品药品分析领域提供一种新的思路。