基于氧化石墨烯荧光传感技术对中药海马的鉴定  

Identification of Traditional Chinese Medicine Seahorses Using Graphene Oxide-based Fluorescent Sensing Technology

作　　者：钟洪金 任九卓林 张灵迂 侯飞侠[1] ZHONG Hongjin;REN Jiuzhuolin;ZHANG Lingyu;HOU Feixia(Pharmacy College,Chengdu University of Traditional Chinese Medicine,State Key Laboratory of Southwestern Chinese Medicine Resources,Chengdu 611137,China)

机构地区：[1]成都中医药大学药学院,西南特色中药资源国家重点实验室,成都611137

出　　处：《中国实验方剂学杂志》2023年第6期185-193,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81903757)。

摘　　要：目的:建立基于氧化石墨烯荧光传感技术鉴定中药海马的方法,为中药鉴定提供一个新的研究思路。方法:在小海马特异性上游引物Ja-F的5’端标记荧光基团FAM作为核酸探针FAM-单链DNA(ssDNA),在下游引物Ja-R的5’端加上RNA聚合酶Ⅱ的识别位点记为Ja-R1,用Ja-F/Ja-R1扩增海马样品,利用体外转录法对扩增产物进行反转录获得小海马ssDNA。取500 nmol·L^(-1)FAM-ssDNA 20μL,90℃孵育5 min,逐渐冷却至室温,加入不同体积的氧化石墨烯溶液(100 mg·L^(-1))和三羟甲基氨基甲烷-盐酸(Tris-HCl)缓冲液(pH 7.6,50 mmol·L^(-1)),使反应终体积为1 mL,混匀,室温避光放置不同时间后测荧光强度,筛选制备传感器的最佳氧化石墨烯浓度和反应时间。再加入探针互补序列FAM-ssDNA-match溶液,室温放置不同时间后测荧光值,筛选荧光恢复的最适反应时间并检测传感器可行性。向核酸探针-氧化石墨烯溶液中分别加入ddH2O和不同浓度的小海马ssDNA进行灵敏度检测,最后根据最佳实验条件对市售海马药材进行检测,验证该方法用于中药材基原鉴定的可行性。结果:构建鉴别小海马的核酸探针-氧化石墨烯生物传感器条件为1 mL反应体积中,10 nmol·L^(-1)FAM-ssDNA+12 mg·L^(-1)氧化石墨烯溶液,室温避光反应20 min,可彻底淬灭探针荧光。传感器可行性检测显示,当向传感器溶液中加入探针互补序列溶液(终浓度90 nmol·L^(-1)),室温反应1 h后,体系的荧光信号明显增强。灵敏度检测显示,该方法能够检测到的小海马ssDNA的最低质量浓度约为10 mg·L^(-1)。将该方法用于检测市售海马药材,发现只有小海马样品有明显的荧光信号。结论:该实验建立的氧化石墨烯荧光传感技术可用于中药海马的基原鉴定。Objective:To establish a method for seahorse identification based on graphene oxide fluorescence sensing technology,and to provide a new research idea for identification of traditional Chinese medicine.Method:The fluorophore FAM was labeled at the 5’end of the specificity upstream primer Ja-F of Hippocampus japonicus as the nucleic acid probe FAM-ssDNA(single strand DNA).The recognition site of RNA polymeraseⅡwas added to its specific downstream primer Ja-R as Ja-R1.The seahorse samples were amplified with Ja-F/Ja-R1 primers,and the ssDNA of H.japonicus was obtained by reverse transcription of the amplification products using vitro transcription method.The 20μL nucleic acid probe FAM-ssDNA(500 nmol·L^(-1))was incubated at 90℃for 5 min,and was gradually cooled to room temperature.Different volume of graphene oxide solution(100 mg·L^(-1))and Tris hydroxymethyl amino methane HCl(Tris-HCl)buffer(50 mmol·L^(-1))were added into each probe solution to make a final reaction volume of 1 mL.The fluorescence intensity of each sample was measured after mixing and placing different times at room temperature away from the light.So that the most appropriate graphene oxide concentration and reaction time were screened for constructing the best nucleic acid probe-graphene oxide biosensor.Adding probe complementary sequence FAM-ssDNA-match solution into the nucleic acid probe-graphene oxide solution,the fluorescence intensity of the reaction mixture was measured after being placed different times at room temperature.Therefore,the optimal reaction time of fluorescence recovery was screened and the feasibility of the sensor was tested.The sensitivity was detected via adding ddH2O as the blank control and different concentration H.japonicus ssDNA into each nucleic acid probe-graphene oxide solution,respectively.Finally,the commercial hippocampal were identified using the optimal experimental condition,and the feasibility of this method for the identification of Chinese medicinal materials was verified.Result:The fluorescenc

关 键 词：氧化石墨烯荧光传感技术 核酸探针 海马 鉴定 单链DNA(ssDNA) 

分 类 号：R284.2[医药卫生—中药学] R289[医药卫生—中医学] R22R2-031R33