A Study Of Electrochemical Biosensors Based On Immobilized GABA Receptor And Rat Small Intestine Tissues

The trouble of falling asleep is perplexing many people,overdrawing people's health,and inducing other disease.Besides,sleep is the important factor of hypertension,diabetes and intelligence development of adolescent.To solve the problem of sleep is the key to treat many other disease,and it's extremely urgent.The sleeping pills we used in clinical is hypnotics,such as diazepam,baclofen and so on.But these drugs could improve the quality of sleep just in a short time,and could cause drug-fast or other untoward effects if the patients take them for a long time.The safety and efficacious drug to replace the hypnotics is the sore to solve the sleeping problem.Seed of wild jujube is the traditional Chinese medicine which can help people have a sedative sleep.Compendium of Materia Medica and Shennong materia medica has recorded it a long time ago.Research shows that the active substance of seed of wild jujube is Jujubaside A and Jujubaside B,of which the amount of Jujubaside A is the most and plays the main role in sedative sleep.Electrochemical biosensor is the delicacy measurement instrument which used electrochemical work station as transducer and bioactive components as sensitive element.Based on this,we used two different bioactive components,that is GABA_BR1 a and rat small intestine tissues,to make two different electrochemical biosensor to realize the test of Jujubaside A.Get the adult SD rat small intestinal tissue,wash up and make them into subsections.Gut and got a small subsection to make a membrane of 0.25 cm².Used the starch sodium-alginate solution as fixative and the microporous membrane to fix onto a glassy carbon electrode for assembling the sandwich-type sensing biosensor.The process of assembled was characterized by Cyclic voltammetry?CV?.With the developed sensor,the response currents induced by s Jujubaside A stimulating corresponding receptors were determined used i-t Chronoamperometry.The results demonstrated that the interaction between Jujubaside A with their receptors were fitting to hyperbola?R²=0.98615?,plotted with method of Lineweaver-Burk,the result of regression is fitting to straight line?R²=0.99002?,and the activation constants were 5.0035×10^-16mol/L.The average number of receptors per cell was calculated as 5,while compared to the bare electrode,the signal amplification factors were 108.The prepared biosensor was continuously?n=10?used to measure 10-5 mol/L Jujubaside A,the relative standard deviation of the measurement results was 4.98%.Five electrochemical tissue sensors prepared in different batches were used to measure the same concentration of Jujubaside A solution.The relative standard deviation of the measurement results?? I/%?was 2.346%,suggesting that the stability of this taste tissue sensor was good.This biosensor?stored over the PBS at 4??was then used tomeasure Jujubaside A of the same concentration every day?n=7?.In the first two days,the change rate of current was almost constant,but was 89.47% of the initial determined value at the third day,and 84.79% of the initial determined value at the seventh day,suggesting that this sensor can be stably stored for at least 7 days.We find GABA receptor gene GABA_BR1 a by the National Center for Biotechnology Information,used eukaryotic expression vector pcDNA3.1,transform DH5? strain and obtain plasmid vector restriction.Liposomal method transfect into HEK-293 T cells,culture and express to obtain the desired protein.Up to that time,we obtained the first bioactive component.We used methods such as self-assembly and others to assemble chitosan,AuNPs,GABA_BR1 a protein solution,thionine-chitosan mixed solution,HRP-AuNPs mixed solution,GABA_BR1 a protein solution and the Bovine Serum Albumin?BSA?which used to block non-specificity locus to the surface of bare glassy carbon electrode in sequence.The process of assembled was characterized by Cyclic voltammetry?CV?.With the developed sensor,the response currents induced by Jujubaside A stimulating corresponding receptors were determined used i-t Chronoamperometry.The results demonstrated that the interaction between Jujubaside A with their receptors were fitting to hyperbola?R²= 97435?,plotted with method of Lineweaver-Burk,the result of regression is fitting to straight line?R² = 0.96416?,and the activation constants were 4.758×10-15mol/L.This just showed the GABA_BR1 a biosensor could simulate the process of signal transmission in the vivo,and revealed the interaction between receptor and ligand.The prepared biosensor was continuously?n=10?used to measure 10-5 mol/L Jujubaside A,the relative standard deviation of the measurement results was 5.86%.Five electrochemical tissue sensors prepared in different batches were used to measure the same concentration of Jujubaside A solution.The relative standard deviation of the measurement results??I/%?was 1.723%,suggesting that the stability of this taste tissue sensor was good.This biosensor?stored over the PBS at 4??was then used to measure Jujubaside A of the same concentration every day?n=7?.In the first two days,the change rate of current was almost constant,but was 92.54% of the initial determined value at the third day,and 88.79% of the initial determined value at the seventh day,suggesting that this sensor can be stably stored for at least 7 days.We discussed interaction between Jujubaside A with its receptors both at the molecular level and the organizationa,though two different electronical biosensors.This provides reference for the evaluation of Jujubaside A and the development and screening of G protein coupled receptor drugs.At the same time,this provides a new idea for the study of the signal transmission,as well as reveals signal amplification factors of cell and average number of receptors per cell.The way of thinking and the parameters such as signal amplification factors of cell and average number of receptors per cell will provide a new direction for further study.