Research On Quality Control Methodology Of Traditional Chinese Medicinal Materials Based On Electrochemical Analysis Technology

There is a classic saying in China,“For Shennong,conscience is the most important thing.For Materia Medica,quality is the foremost thing”.As an important traditional cultural treasure in China,traditional Chinese medicine is rich in resources and has a long history for five thousands.It has its own unique theory and valuable experience.In recent years,traditional Chinese medicine is favored and valued by many countries all over the world owing to its unique curative effect.At the same time,the quality control of traditional Chinese medicine has also received more and more attention.For a long time,the traditional analysis method of the traditional Chinese medicine has restricted its application and the development in the field of the quality of traditional Chinese medicine at a certain extent due to its defects in sensitivity or accuracy,which is also one of the obstacles to restrict traditional Chinese medicine towards its internalization.In order to make traditional Chinese medicine international and be truly accepted by the world,we must strive to improve the quality of traditional Chinese medicine and raise the standards of the quality control of traditional Chinese medicine.Developing simple,convenient and efficient quality control technology of traditional Chinese medicinal materials has become a hot issue for many scientists to carry out the quality control research of traditional Chinese medicine.Compared with the traditional method,the electrochemical analysis method shows many advantages such as low cost,simple operation,simple pretreatment,fast response,low energy consumption,tiny apparatus,being suitable for online analysis on the spot,etc.The research and application of developing quality detection method of traditional Chinese medicinal materials on the spot are getting more and more attention.And the electrochemical analysis method is also encouraged in the quality control research of traditional Chinese medicine.The research is to carry out a systematic study on the quality control research of traditional Chinese medicine by utilizing electrochemical analysis technology.It has the following four steps’research work I the aspect of the quality control of the traditional Chinese medicine.Firstly,two types of new electrochemical DNAsensors are designed,which generate electrochemical response signals through host-guest recognition ofβ-cyclodextrin（β--CD）and tetraferrocene.And some means such as electroactive probes and catalytic hairpin self-assembly signal are adopted to improve the response signal strength of the electrochemical biosensors so as to achieve the goal of gene identification for precious Chinese medicinal materials such as“Cordyceps Sinensis”.Secondly,basing on the principle of dual signal amplification of DNA walker and tetraferrocene,we construct the electrochemical biosensors and apply them to the quantitative determination of heavy metal“Hg²⁺”in Chinese medicinal materials“Cordyceps Sinensis”.Thirdly,we utilize electrochemical method to carry out the content determination of Rhein in Rhubarb.Finally,we conduct a systematic research about the methodology of utilizingmethod of DPV to determine the content of Rhein in Rhubarb.The specific research results are as follows:（1）The research on the Construction of electrochemical DNA sensor based on the host-guest recognition ofβ-CD and new tetraferrocene compound and the identification for Cordyceps sinensisIn this part,we first design and synthesize a new tetraferrocene compound,on this basis,we design and prepare a new electrochemical DNA sensor based on the tetraferrocene andβ-cyclodextrin which are used as electrochemical markers,and this electrochemical biosensor can generate and amplify the electrochemical signal strength of DPV by using host-guest recognition ofβ-CD and new tetraferrocene compound.At first,we select nucleic acid aptamer probe with stem ring structure and use a marker of tetraferrocene at 3 and 5 ends.Close loop structure and use a marker of tetraferrocene compound from reaching the electrode surface.Then,stem loop structure is opened after the gene processing of Cordyceps Sinensis.Therefore,tetraferrocene compound can be quickly attached to the electrode through host-guestrecognition.Subsequently,electrochemicalsignalof tetraferrocene compound can be measured.The hybrization process constructed by this method is directly carried out in the solution,has no use for fixing the probe in advance and avoids unnecessary electrode modification.The detection limit of this designed electrochemical biosensor can be as low as 1.5×10^-12M.（2）The research on the construction of electrochemical DNA sensor based on the strategy of dual signal amplification by combining target circulation CHA with host-guest recognition between betweenβ-CD and new tetraferrocene compound and its identification of Cordyceps sinensisIn this part,We optimized the sensor constructed in the first part,on the basis of target cyclic CHA strategy,we construct a new electrochemical DNA sensor with dou ble signal amplification strategy based on the host-guest recognition effect between β-CD and a new type of tetraferrocene compound.And his type of electrochemical DNA sensor highly efficient amplified electrochemical signals through a dual signal amplification strategy in the electrochemical detecti-on process.This biosensor utilizes the host-guest recognition between tetraferrocene compound andβ-CD to connect tetraferrocene-DNA andβ-CD-Au E.Target nucleic acid fragments are recyled owing to the hairpin.As a result,response signal of the biosensor is greatly improved.This biosensor can detect Cordyceps Sinensis gene hypersensitively.Its lowest detection limit is 0.17 p M.The research provides an effective method for detecting Cordyceps Sinensis,with characteristics of low background signal,low detection limit,simple equipment and other characteristics.（3）The research on the construction of electrochemical biosensor based on the strategy of DNA Walker and new tetraferrocene compound by dual signal amplification and the determination of Hg²⁺in Cordyceps sinensisIn this part,we construct a highly integrated electrochemical biosensor which is based on the double signal amplification strategy combined DNA Walker with tetrafe-rrocene.The electrochemical biosensor which we construct is used to determine the heavy metal ion“Hg²⁺”in Chinese medicinal materials Cordyceps Sinensis.Since divalent mercury ions can form stable thymidine-divalent mercury ions-thymidine“T-Hg²⁺-T”base pairs,we introduce an auxiliary DNA.The auxiliary DNA can have hybridization reaction with chains efficiently,thus causing Hg²⁺to trigger DNA enzyme walking chain then cut off the substrate chain marked by tetraferrocene automatically and continuously.At the same time,owing to DNA Walker strategy,one Hg²⁺can cause multiple DPV responses leading to a significant decline in the intensity of electrochemical response signal.Under this condition,Hg²⁺presents a good liner relationship within the concentration range of 0.1p M～0.195μM and the detection limit can reach to 4.8 f M.It can work normally,even when dealing with Chinese medicinal materials complex samples such as Cordyceps Sinensis.The rate of recovery for Hg²⁺is 97～103%,its total average rate of recovery is 99.49%,and all the relative standard deviation RSD is less than 3.0%.The electrochemical biosensor platform doesn’t require regent,being self-generating,highly sensitive and easy to operate.It provides broad technology and application prospects for detection Hg²⁺in the environment and samples of Chinese medicinal materials.（4）The research on the methodology for the content determination of Rhein in Rhubarb based on different pulse voltammatry of electrochemical technologyIn this part,we establish a simple,rapid and sensitive method for determining content of the Rhein in Chinses medicine Rhubarb by DPV of electrochemical technology.The electrochemical behavior of Rhein on the glass carbon electrod is also studied.We investigate several extraction methods such as extraction method,boiling method,and ultrasonic method to obtain their respective extration efficiency of Rhein from Chinese medicine Rhubarb,and we also examine the impact of extraction efficiency at different extraction time.We also adopt DPV to determine the content Rhein in the Rhubarb extracting solution obtained from the best extraction method.At the same time,we compare the determination result of the electrochemical method with the result which is obtained by using HPLC to determine the content of Rhein in the extracting solution.By comparing determination results of electrochemical method and HPLC,the best detection condition of the electrochemical method for the determination of the Rhein in Chinese medicine Rhubarb and the best extraction method of Rhein are obtained,and we also confirm the advantages of the electrochemical method in determining the content of the active ingredient of traditional Chinses medicine.The best extraction method of Rheinfrom Rhubarb is 95%ethanol extraction for 50～60 min;and under the the best determination condition,Rhein has a good linear relationship in the range of0.0270～0.0470 mg/ml,Rehin’s correlation coefficient r²is 0.9992,and its rate of recovery is 97～105%.And all the relative standard deviation RSD is less than 4%.The experiments show that the electrochemical method presents the advantages of high selectivity and high accuracy.The research findings of this paper can establish the rapid identification method based on the principle of the electrochemical gene identification sensor of traditional Chinese Medicinal materials and succeed in establishing the electrochemical sensing method for detecting the harmful substances of traditional Chinese Medicinal materials.Moreover,the findings can be lay a foundation for the establishment of electrochemical quantitative analysis method of active ingredients in traditional Chinese medicinal matrials.So then,the accuracy for the results of quality control of traditional Chinses medicine by modern science and technology can bu improved to the maximum.On the whole,these findings provide powerful,scientific and technical support for achieving the modernization of quality control of traditional Chinses medicine.As a result,the research findings also provide new ideas for the application of electrochemical analysis method to the quality control research of traditional Chinses medicinal materials.