| Medicinal plants are the important source of natural medicine and play an important role in the development of Chinese traditional medicine.The resources of medicinal plants in our country are rich and various,but at the same time their sources are relatively complex so that the quality are discrepant.Therefore,it is vital to regulate the quality of medicinal plants.Taking Chinese Pharmacopoeia as an example,a series of drug quality control standards have been formulated in China.The analysis methods involved in quality control are accurate and reliable,but they are stuck in the in vitro detection due to deficiencies such as the need to destroy plant tissues,tedious pre-processing steps,and expensive dependence on equipments.In fact,the content and composition of active ingredients in medicinal plants alwawys change with the growth season,years and environment.Even in different cells,tissues and organs of the same medicinal plant,they may have different medicinal properties at different stages of development.In vitro detection methods usually ignore the parameters involved in biological environment,which may lead to high or low detection results.Therefore,it is necessary to develop a point-of-care testing method.In this study,DNA molecular identification and active component content analysis in quality control projects were used as the work objects.A reagentless electrochemical sensor based on three-strand complex and a portable electrochemical sensor based on screen printing electrode were proposed respectively.Catalytic hairpin reactions,novel signal markers and inorganic-organic materialsb were used to improve the detection performance of the sensors.The constructed sensors were applied to the detection of DNA and total flavonoids content in medicinal plants,and the specific research contents are as follows:（1）ITS 2 sequence of Bupleurum was selected as target DNA by searching Gen Bank database,and tetraferrocene synthesized previously by our research group was used as probe signal marker.Hairpin probes were designed according to the principle of base complementary pairing,and the two hairpin probes were connected into a three-strand complex using a connecting chain.The complex was assembled on the surface of gold electrode through Au-S bond,and a reagentless electrochemical sensor was constructed for the rapid detection of Bupleurum DNA.The rationality of the three-chain complex was verified by fluorescence and gel electrophoresis.DPV method was used to detect the target DNA,and it was shown that the current change value presented a good linear relationship with the target DNA concentration in the range of 1×10-13M～1×10-9M,and the working curve equation was I=-0.3019Lg C-4.2443（R2=0.9966）.The detection limit（LOD）was calculated as 0.085p M.In addition,the constructed sensor has good specificity,repeatability,and has been successfully used in the detection of PCR products of Bupleurum.The detection process does not need to add exogenous reagents,which improves the detection efficiency and lays a foundation for rapid detection.（2）The green,non-toxic and biocompatibleβ-CD-MOF was synthesized by steam diffusion method,and deposited on the well dispersed r GO.The PVP-r GO/β-CD-MOF composite was synthesized by deposition on the well dispersed r GO.And a portable electrochemical sensor was prepared by modifying composite material on the surface of screen printed electrode by drip coating method.Using rutin as a standard product,the electrochemical response of rutin with different concentrations in PVP-r GO/β-CD-MOF/SPE was investigated by DPV method.The results show that in the range of 6.4×10-7M～8×10-5M,there is a good linear relationship between the current and the logarithm of rutin concentration.The standard working curve equation is I=5.60741Lg C+35.61128,R2=0.99826,and LOD is 2.20281×10-8M.The content of flavonoids in stems and leaves of fresh Houttuynia Cordata was 363.2μM rutin·g-1and 1234.2μM rutin·g-1,respectively.The content of total flavonoids in leaf extract of H.Cordata was significantly higher than that in stem extract,which was consistent with the results reported in the literature.In order to contrast,rutin was detected by ultraviolet spectrophotometry.In addition,the contents of flavonoids in stems and leaves of Houttuynia Cordata were detected in situ using portable sensors.Compared with traditional electrochemical sensors,the reagentless electrochemical sensor prepared in this paper significantly improves the detection sensitivity in the process of detecting target DNA by combining the catalytic hairpin reaction and the tetraferrocene signal amplification strategy.Meanwhile,the construction of the three-chain complex eliminates the step of adding exogenous reagents in the detection process and improves the detection efficiency,which shows high application potential.The portable sensor prepared in this paper shows a wider linear range,lower detection limit and shorter detection time than the ultraviolet spectrophotometry,and has good anti-interference ability,stability and repeatability.In addition,there is no need to pre-treat the samples in the detection process and can achieve the point-of-care testing,which is of great significance in monitoring,controlling and even intervening the content of active ingredients in the growth process of medicinal plants. |
PDF Full Text Request