Preparation Of Nano-sensing Needle And Its Applications In Monitoring Bioactive Molecules And Nitric Oxide Release Regulated By Traditional Chinese Medicine

Objectives:Traditional Chinese medicine(TCM)is famous for its holistic therapy and scientific theoretical system.Although TCM accumulates rich experience in the etiology,pathogenesis and treatment of various diseases,the mechanism of TCM is yet understood fully.According to the theory of TCM,for different symptoms,using different TCM prescription can make the whole brain or different levels of brain neurotransmitters and its metabolites level change in animal models.These changes may have differences in degree,property and mechanism,but TCM plays an important role in regulating neurotransmitters and its metabolites.Similarly,acupuncture,originated in China,is also the treasure of our country with a long history.Different from the drugs intervention,acupuncture maybe indirectly play its role by inspiring the body's potential ability and activated a variety of regulatory mechanism.Although acupuncture holds the functions of therapeutic aims,the mechanism of the acupuncture medicine remains unclear.It is believed that a molecular event usually occurs in the three-dimensional structure of acupoints and meridians,while acupuncture needles are manipulated on the body.Therefore,it is of great significance to develop a unique sensing needle for measuring neurotransmitter levels and molecular events and figure out the relatedmolecular events in acupuncture medicine and TCM therapy.Using acupuncture needle as substrate,combined with multiple modern technology,the sensing needle owns the ability of sense pH,oxygen pressure,temperature,histamine,calcium ion and so on in the human body with Chinese characteristics.In early studies,they often explored the polymer affinity coating for monitoring the signal molecules.Apparently,the modification is carried out through physical adsorption,resulting in uncontrollable membrane thickness.More importantly,the polymer is easy to peel off from the needle body while the acupuncture needles are inserted into the body.Both rough sensing surface and unstable infrastructure make the sensing needles incapable in terms of reproducibility and stability in practical applications.So there is a demand for preparation of novel acupuncture needles with a stable and sensitive ability.Nanomaterials have been widely studied by researchers in recent years for its special properties,such as large specific surface area,strong adsorption capacity,high catalytic efficiency,high surface activity site,and so on.Graphene,as a typical representative among them with its large specific surface area,high conductivity,larger adsorption performance and faster electron transfer rate and excellent electric catalytic activity,has become the ideal material for preparation of electrochemical sensors and biosensors.And graphene has gotten widely attention in the development of energy storage materials,biological device design,biological molecular detection,biochemical analysis and molecular medical diagnosis.In this report,we develop a unique nanomaterials functionalized sensing needle,which combined nanotechnology and the traditional acupuncture needle.The functionalized sensing needle can be applied in monitoring bioactive molecules(such as dopamine(DA)and nitric oxide(NO))and the release of NO regulated by TCM.This functionalized sensing needle wasapplied in realizing real-time detection of the signal molecule in vivo,which not only can reflect the neurotransmitter effect regulated by TCM,but also can explore the change of the molecular effect in the needle-insert process,providing theoretical and technical support for the mechanism of TCM or acupuncture therapy.Methods and Results:In this work,a novel graphene or graphene-functionalized sensing needle biosensor have been developed.Based on this,the electrochemical detection were realized on pH,DA and real-time monitor NO release in acupoints.What's more,the regulation of different TCM on the NO release was investigated,and the nano-sensing needle was used to detect NO release regulated by four kinds of TCM active ingredients.The main contents of this topic include the following three parts.Part I: Preparation of graphene-modified sensing needle and its application in detecting neurotransmitters dopaminePrimarily,GO was prepared by modified Hummers methods and Raman spectroscope was used to characterize it.Then One-step electrochemical reduced graphene oxide(ERGO)was fabricated and immobilized onto the tip of acupuncture needle(G-AN).What's more,in order to study its electrochemical behavior,the redox probe [Fe(CN)6]3-/4-was used to characterize the bare acupuncture needle and nanomaterial modified acupuncture needle.The results exhibited that GO has been synthesized successfully and the redox peak current was larger on the G-AN than bare acupuncture needle,which indicate that ERGO can improve the electron transfer rate on the sensor surface.The G-AN's pH dependence was measured by recording the open circuit potentials in various PBS buffer solutions,ranging from p H 2.0 to 10.0.It was observed that the signal stabilized within a few seconds and kept stable duringthe measured time.The potential signals decreased when the value of pH were increased.The excellent performance was also showed in terms of linearity(E=0.0209-0.0299 pH,R2 = 0.9921).The above-mentioned linear equation exhibited that the sensitivity of linear pH response was 29.9 mV/pH.Finally,we use the G-AN for electrochemical detection of DA.The G-AN exhibited good selectivity in the presence of AA and DA.Under the optimal condtions,the G-AN was further used for quantitative analysis of DA,and the oxidation peak current of DA and its concentration showed a good linear relationship in the range of 1.0×10-7 mol/L~1.0×10-5 mol/L,with a limit of detection of 0.24 ?M.The as-prepared G-AN was immersed in PBS solution for one week and inserted into brain tissue for stability test,SEM showes that the modified layers of G-AN are stable.The practical applications of the G-AN in serum sample were successfully conducted by the standard addition method.Part II: A sensitive acupuncture needle microsensor for real-time monitoring of nitric oxide in acupoints of ratsFirstly,iron-porphyrin functionalized graphene composite(FGPC)was prepared by hydrothermal synthesis,and then scanning electron microscopy(SEM),ultraviolet spectroscopy and X ray energy spectrum were used to characterize the FGPC.Sencondly,A gold film(AuNPs)is sprayed onto the tip of acupuncture needle by sputter coater.Subsequently,the electrochemical deposition method is employed to deposit FGPC on the tip surface,while the other part of needle was coated with insulation paste.We develop a unique acupuncture needle-based NO microsensor,capable of real-time monitoring NO in acupoint of rats by utilizing the advantages of electrical performance of graphene and the great catalytic property of porphyrin.In PBS solution,FGPC acted as the excellent catalytic capability and the FGPC/AN showed larger current response than others.This attributes to thefact that FeTCP,simulating the enzyme active site,could significantly enhance the catalytic capability to NO,but a negligible response to other interfering substances.Moreover,RGO can facilitate the fast electron-transfer between FeTCP and acupuncture needle.Under the optimal condtions,the FGPC/AN was further used for quantitative analysis of NO,and the oxidation peak current of NO and its concentration showed a good linear relationship in the range of 5.0×10-9 mol/L~2×10-7 mol/L,with a limit of detection of 3.2 n M.After immersed in 1M KCl solution for 10 days,the current signal of FGPC/AN remained more than 82% of the initial signal,showes that FGPC/AN has good stability.In order to test the sensor's capability of detecting NO in vivo,real-time detection of NO on HUVECs at cell level was conducted firstly.When L-Arg was introduced into the cells,the current increased remarkably and then started to stabilize slowly.On the contrary,introduction of L-NAME diminished the current change,owing to the specific inhibiting behavior of L-NAME toward NO release.A control experiment was conducted to verify that the increasing current was caused by the NO release from HUVECs.When the same concentration of L-Arg was added into the detection chamber without cells,no obvious current response was observed.All above experiments demonstrate that the FGPC/AN is capable of monitoring NO in real time at cell level.On the basis of the excellent properties of the FGPC modified needle,the needle was then inserted into the different acupoints to realize real-time monitoring of NO dynamic change while stimulated by L-Arg.The typical acupoints of zusanli(ST36),Zhongwan(CV12)and Quchi(LI11)were selected,respectively.After the FGPC/AN was inserted into the acupoint,followed by L-Arg stimulation,an apparent current increase was observed.As a control,if stimulated by PBS solution instead of L-Arg,hardly currentchange was seen,indicating that the FGPC/AN could realize the real-time monitoring of NO in acupoints.Then,real-time monitoring of NO in other parts was also carried out,and the similar results were obtained.All these results indicate a direct real-time measurement of NO in acupoints have been realized,demonstrating the needle's superior detection capability in vivo.Part III: TCM active ingredients affected the NO release at cell levelAbnormal NO concentration in the body may lead to serious diseases occurrence and development.So it is of great significance to regulate NO physiological level by TCM.We choose four kinds of single TCM for the next experiments.There are sinomenium acutum,leonurus japonicas houtt,pueraria lobata and allium sativum,and the main active ingredient are sinomenine,stachydrine,puerarin and allicin,respectively.In order to observe the influence of TCM on cell activity,human umbilical vein endothelial cells(HUVECs)are cultivated in 96-well plates with different concentrations of drugs for 24 h.Then MTT method was used to measure the cell activity.The experimental result indicated that the four active ingredients had no obvious toxic effect on endothelial cells in the concentration of 50 ?g/mL or lower.In order to detect the NO release level in HUVECs,drugs were cultured with cells,and NO commercial kit was also used.Through the experiment data,it was seen that sinomenine and stachydrine inhibited NO release in cells,and puerarin and allicin promoted NO release in cells.Furthermore,FGPC/AN was used to monitor NO release regulated by TCM by electrochemical methods.The FGPC/AN was put into cell plates without TCM,followed by L-Arg stimulation,an apparent current increase was observed.Through the experiment data,it was seen that sinomenine and stachydrine inhibited NO release in cells,results show that current of stachydrine and sinomenine were obviously less than control group.And puerarin and allicin promoted NO release in cells,results show that current ofpuerarin and allicin were more than control group.The detection result of the FGPC/AN is consistent with that of the NO commercial kit.Conclusion:1.We have developed a unique nanomaterials functionalized sensing needle,which combined nanotechnology and the traditional acupuncture needle.2.The prepared G-AN showed a superior stability,high selectivity and high sensitivity.3.The G-AN's pH dependence was measured by recording the open circuit potential,and its excellent performance was achieved.What's more,the G-AN was further used for detection of DA and was also successfully used to test DA in human serum samples.4.The FGPC functionalized acupuncture needle exhibited efficient electrocatalysis,high sensitivity and good selectivity toward NO, making it capable of realizing real-time monitoring of NO in complex environments including cell measurement and in vivo detection.5.The TCM active ingredients can regulate NO release.Stachydrine and sinomenine could inhibit the release of NO.On the other side,puerarin and allicin could promote the release of NO.