Study On Aptasensor Based On A Nanohybrid Material Composed Of Reduced Graphene Oxide And A Metal-organic Framework For ESAT-6 Ultrasensitive Detection

Tuberculosis has been a public health problem that has seriously threatened human life since ancient times.It is a chronic respiratory infection caused by Mycobacterium tuberculosis(MTB)infection,which has become the first cause of death in infectious diseases.As of now,about one-third of the world's population has been or is suffering from MTB.According to the World Health Organization(WHO),the number of new tuberculosis patients worldwide reached 10.4 million in 2015,and tuberculosis has caused more than 1.4 million deaths.With the irregular treatment leading to the increase of drug-resistant tuberculosis strains,the accelerationofpopulationmovementsandtheincreasein AIDS-contaminated tuberculosis patients,the tuberculosis epidemic has spread.Early,timely and accurate detection of MTB is an extricable approach to effectively control tuberculosis,reduce the prevalent of tuberculosis and the emergence of drug-resistant strains.At present,the frequently used MTB detection methods in clinical practice include“gold standard”for tuberculosis diagnosis bacterial culture,polymerase chain reaction(PCR)and enzyme-linked immunosorbent assay(ELISA).These approaches possess different degrees of defects,such as long-time consuming,poor stability,high false positive rate,many influencing factors,poor sensitivity and specificity.In addition,Bacille Calmette-Guerin(BCG)vaccination is widely carried out in China,which makes it more difficult to accurately detect MTB by avoiding false positive results caused by BCG.When MTB infects the host,the specific antigen of MTB will appear firstly in the patient.These antigens are not affected by the immune status of the patient.Therefore,the detection of MTB antigen in body fluid has significance for the early diagnosis of tuberculosis.The6-kDa early secretory antigenic target(ESAT-6)is an early secreted protein produced by MTB,which is only found in pathogenic mycobacteria,but is absent in BCG.It is an ideal biomarker for MTB determination.The aptamer has the advantages of simple synthesis,low cost,stable nature,easy modification,wide applicability and the ability to specifically bind to the target ligand.It has a higher affinity for binding to the target than the antibody.The electrochemical aptasensor constructed using the aptamer as the recognition element has both high specificity of the aptamer and fast response and high sensitivity of the electrochemical analysis method.In order to overcome the shortcomings of the MTB detection means,this study utilized the poly(diallyldimethylammonium chloride)functioned reducedgrapheneoxidedopedwithmetal-organicframework(P-MOF-rGO)nanohybrid with large specific surface area,high conductivity and stability to construct an electrochemical aptasensor for MTB detection to achieve early and accurate diagnosis of tuberculosis.The sensor uses ESAT-6 as a detection target and its specific aptamer as a recognition element.The main contents of this study are as follows:Objective:The P-MOF-rGO nanocomposite was employed as the immobilized material of the electrode to fabricate an label-free electrochemical aptasensor for the ultrasensitive detection of tuberculosis antigen ESAT-6,which provided an effective platform for early and accurate diagnosis of tuberculosis.Methods:The P-MOF-rGO nanohybrid was prepared and fixed on the surface of a glassy carbon electrode(GCE).The Toluidine Blue(TB)was incubated on the electrode as an electroactive substance,and platinum/gold core/shell(Pt@Au)was employed for electrical signal amplification.The thiolated ESAT-6 binding aptamer(EBA)was bound to the GCE surface by Au-S bond,and the excess active site was blocked with bovine serum albumin(BSA).After constructing the aptasensor,the volume of TB and Pt@Au was optimized.The sensitivity of the sensor was determined by measuring the change in current between the aptamer and the different concentrations of ESAT-6 before and after binding.The specificity was examined by using endogenous molecules such as glucose,BSA,ascorbic acid(AA),and 10-kDa culture filtrate protein(CFP-10)as interfering substances.To investigate the stability of the aptasensor,the electrode was taken successive 50 cyclic scans with cyclic voltammetry(CV).Five electrodes incubated with the same concentration of ESAT-6(1 ng/mL)were tested to examine the reproducibility of the sensor.ESAT-6 in serum samples was analyzed to evaluate the clinical applicability of the sensor.Results:The constructed aptasensor has the optimal detection behavior when the TB volume is 5?L and the Pt@Au volume is 8?L.The concentration of ESAT-6 was linearly related to the current response in the range of 1.0×10~-44 ng/mL~2.0×10~2 ng/mL.The limit of detection(LOD)of the sensor was 3.3×10~-55 ng/mL.The results of interference testshowed that the sensor can specifically recognize ESAT-6 and hold poor response to interferents.The stability test indicated that the sensor possessed good stability.After 50 consecutive scans,the current response only dropped by about 3.5%.According to the results of the reproducibility test,sensor with a good reproducibility,the relative standard deviation(RSD)of the five electrodes was 2.9%.When the aptasensor detected ESAT-6 in serum samples,the spiked recovery rate was 99%~102%,and the results was satisfactory.Conclusions:The electrochemical aptasensor established for ESAT-6ultrasensitive detection is simple to prepare,has a wide linear range,low detection limit,excellent stability and reproducibility.Determination of ESAT-6 in actual serum samples demonstrate good specificity and satisfactory spike recovery,which is promising to provide an effective technical platform for timely and accurate diagnosis of MTB infection.