| Dopamine(DA)is a catecholamine neurotransmitter that widely exists in the central nervous system.It plays an important role in the nervous system,cardiovascular system,renal system,and regulates various physiological activities.DA is stored in the synaptic vesicles.When dopaminergic neurons are stimulated,DA is released by exocytosis,which acts on the target cells with dopamine receptors on the cell membrane,and then transmits the intercellular signals to regulate the corresponding physiological functions.This neurotransmitter plays an important role in maintaining normal brain activity and function.Abnormal levels of DA are directly related to various neurological disorders such as Parkinson’s disease,schizophrenia and attention deficit hyperactivity disorders.Therefore,rapid detection of DA levels in the brain is of great significance for biomedical diagnosis.In recent decades,field effect transistor(FET)biosensors have been regarded as one of the most promising biosensors because of its high sensitivity,good specificity,low cost,labeling free,easy integration and miniaturization.With the development of nanotechnology,nano-materials such as: silicon nanowires,carbon nanotubes,graphene,molybdenum disulfide,etc are widely used in FET sensors due to its large specific surface area,good chemical stability,proven biocompatibility and environment friendly,which significantly improve the stability and sensitivity of biosensors,reduce analysis time and amplify detection signals.At present,it has been reported that an ultra-sensitive FET biosensor could real-time monitor the DA released from cells,and the detection limit was as low as 10 p M.However,FET biosensors are mostly planar chips now,which are too large to be used for in vivo detection.Real-time monitoring of the dynamics of DA molecules in the brain is an important content to study the molecular basis of diseases.Based on this purpose,this paper aims to fabricate a needle-shaped FET biosensor,which can be applied to the label-free and highly sensitive detection of DA,and further applied to the in vivo detection of dopamine in rat brain.The specific work is as follows:Part Ⅰ: Fabrication of graphene needle-shaped field effect transistor biosensorA medical acupuncture needle which is easy to penetrate the body was selected as the substrate of the sensor,and the surface of acupuncture needles was coated subsequently by Parylene insulating layer,Au layer and Parylene insulating layer using technologies of vacuum gas phase deposition and electroplating,to form the structure of Parylene-Au-Parylene three-layer film covering the surface of the acupuncture needle.A mcropipette beveler was employed to grind the tip of a needle to get a smooth cross section of the tip,and expose the structure of stainless steel-parylene-Au-parylene layer inside.The stainless steel and Au layers are the source and drain electrodes of FET devices,respectively.The Parylene layer sandwiched between them is the insulation channel.Then reduced graphene oxide(RGO)prepared by chemical reduction method was dropped on a cross section of the tip to connect stainless steel and Au film,and the preparation of the needle-shaped RGO-FET was completed.Subsequently,a series of electrical measurements were carried out on the needle-shaped FET device,and ambipolar characteristics of graphene were demonstrated.In order to evaluate the response of the sensor to charge changes,the measurements of the solution p H value were conducted,and the results showed a good response to hydrogen ions in the solution with a sensitivity of 37 m V/p H.Then,synthetic glutamate receptors were modified on the RGO channel of the sensing interface.When glutamate was present in the solution,glutamate receptors could specifically capture glutamate,and the negative charge of glutamate produced n-type doping on the RGO channel,thus causing Dirac Point of transfer curve negatively shifted and producing a specific response to glutamate.The experimental results show that needle-shaped RGO-FET in this paper has good electrical properties.By modifying specific biological recognition molecules on the channel,the needle-shaped biosensor also can response specifically to the target which demonstrated that the needle-shaped RGO-FET sensor was successfully prepared.Part Ⅱ: Monitor dopamine dynamics in rat brain with needle-shaped RGO-FET biosensorIn this part,based on needle-shaped RGO-FET devices prepared above,a DA sensor response specifically to DA was constructed and successfully applied to real-time monitor the dynamics of DA in rat brain.An amino aptamer against DA was immobilized on the surface of RGO channel by 1-Pyrenebutanoic acid succinimidyl ester(PASE)linker.When DA was present in the buffer solution,Aptamer could specifically bind with DA and undergo conformational changes,which led to n-type doping effect to RGO by charge transfer,which eventually led to decline in current,and then the DA can be sensed.The experimental results showed that the sensor could still maintain good selectivity to DA in the presence of other neurotransmitters.The sensor had a good linear response to DA in the concentration range of 1 n M to 10 μM,and the detection limit was as low as 1 n M.The sensor was then placed in the striatum,a DA-rich region of the rat brain,for real-time monitoring DA in vivo.Using high potassium stimulant to promote the release of DA from neurons,the results showed that the sensor produced a significant electrical response to the released DA.In addition,drugs of Nomifensine and Pimozide were used to regulate the release of DA in the brain of rats,and then electrical measurements were conducted.Nomifensine can increase the concentration of DA in extracellular fluid by inhibiting DA transport,while the antagonism of Pimozide can inhibit the generation and transport of DA vesicles,thus reducing the release of DA.The results of electrical measurements showed that the current variations in the Nomifensine groups were significantly greater than that in the physiological control groups,while the current variations in the Pimozide groups were less than that in the physiological control groups,indicating that the sensor was sensitive enough to distinguish the drug’s regulation of DA release.Due to the direct relationship between abnormal DA release and Parkinson’s disease,this paper further prepared a Parkinsonian Rat Model.The real-time mearurements were carried out by using sensors to monitor the electrical responses generated by released DA in striatum under high potassium stimulant and compared with electrical responses measured in normal rats.The dopamine nervous system of Parkinson’s rats was damaged by 6-hydroxydopamine,and the ability to synthesize and release DA were weakened.The in vivo detection results shown that the electrical signals measured in the Parkinson’s model group were significantly lower than those in the normal control group,which was consistent with the literature reported.The above experimental results confirmed that the needle-shaped FET biosensor prepared based on acupuncture needle in this paper was successful.It can be applied to real-time monitor the dynamic changes of DA in the rat brain under the stimulation of high potassium solution.Our fabricated need-shaped FET biosensor provide a new method for in vivo detection. |
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