Identification of Traditional Chinese Medicine Seahorses Using Graphene Oxide-based Fluorescent Sensing Technology / 中国实验方剂学杂志

ABSTRACT

ObjectiveTo 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. MethodThe 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 polymerase Ⅱ was 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 20 μL 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. ResultThe fluorescence of 1 mL reaction mixture including 10 nmol·L-1 nucleic acid probe FAM-ssDNA and 12 mg·L-1 go solution for 20 min at room temperature away from the light could be completely quenched. Feasibility test of the biosensor showed that when probe complementary sequence FAM-ssDNA-match solution （final concentration 90 nmol·L-1） was added to the biosensor solution and reacted 1 h reaction at room temperature， the fluorescence signal was significantly enhanced. Sensitivity test showed that the minimum concentration of ssDNA detected by this method was about 10 mg·L-1. This method was used to detect commercial seahorses， and only H. japonicus samples had obvious fluorescence signal. ConclusionThe graphene oxide-based fluorescent sensing technology could be used for zoological origin survey of commercial hippocampus.