A New Biosensor Based On The Synergistic Amplification Strategy Of GO@Fe₃O₄@Pt Nanocomposite And Rolling Circle Amplification For Rapid Detection Of Mycobacterium Tuberculosis Secretory Protein MPT64

ObjectiveTuberculosis caused by Mycobacterium tuberculosis（Mtb）is an infectious disease with high morbidity and mortality.The traditional detectionmethodsforMtbhaveunsatisfactorysensitivityor time-consuming,which cannot meet the clinical requirements.Therefore,it is urgent to research a new method to provide reliable laboratorial evidence for early and rapid diagnosis of Mtb infection in clinic.In this study,a novel electrochemical biosensor is going to be constructed based on a synergistic amplification strategy of GO@Fe₃O₄@Pt nanocomposite and rolling circle amplification to achieve ultrasensitive and rapid detection of Mtb secretory protein MPT64 in blood.After the optimization of the key parameters,the analytical performance of the new biosensor is evaluated.Methods1.Synthesis of GO@Fe₃O₄@Pt nanocomposite:firstly,we synthesised GO@Fe₃O₄ with amidation reaction mediated by EDC/NHS;and then the GO@Fe₃O₄@Pt nanocomposite was synthesized by in situ reduction of Pt⁴⁺using NaBH₄.The morphology and chemical composition of GO@Fe₃O₄@Pt nanocomposite was characterized by TEM and EDS,respectively.2.Preparation of the circular DNA:the primer and phosphorylated padlock probe were hybridized by heating at 95°C for 5 min and cooling down to 22°C gradually.The circular DNA was synthesized with the help of T4 DNA ligase at 22°C for overnight.The single-stranded DNA and mismatching double-stranded DNA were digested by Exonuclease I and Exonuclease III.The circular DNA was purified by ethanol precipitation.Agarose gel electrophoresis assay was used to validate the synthesis of circular DNA.3.Fabrication of the biosensor:protein G was fixated on the gold electrode by Au-histidine bond.Capture antibody was fixated on the gold electrode by connecting its Fc region with protein G.BSA was applied to block the gold electrode.With the existence of MPT64,aptamer-primer can be bonded to the surface of gold electrode,it can initiate rolling circle amplification to obtain long single-stranded DNA.GO@Fe₃O₄@Pt nanocomposite was adsorbed the single-stranded DNA byπ-πbond,and it can catalyze the decomposition of H₂O₂.And then the electrochemical signal was generated and the quantitative detection of MPT64 was achieved.4.Optimizationofexperimentalconditions:thespecific aptamer-primer toward MPT64 was chosen from two candidate.The key parameters were optimized for the higher signal to noise ratio of the biosensor,including the concentrations of protein G,capture antibody,aptamer-primer,the reaction time of rolling circle amplification,and the pH of working solution.5.Analytical performance evaluation of biosensor:the repeatability,specificity,linear range,limit of detection and stability of the biosensor were evaluated by measuring different concentrations of MPT64.6.Recovery test:MPT64 protein was added to the serum for preparing different concentrations of MPT64 serum solution.And then they were detected by the biosensor,respectively.Results1.Characterization of GO@Fe₃O₄@Pt nanocomposite:TEM results indicated that Fe₃O₄ and Pt nanoparticles were anchored on the surface of GO.EDS confirmed the presence of C,O,Fe,Pt in GO@Fe₃O₄@Pt nanocomposite.2.The results of rolling circle amplification:Agarose gel electrophoresis assay revealed that the electrophoretic bands increased and the position of the main band was shifted after T4 DNA ligase added.But only the circular DNA was existed after being treated with Exonuclease I and Exonuclease III.Large molecular weight products were observed after rolling circle amplification reaction,its electrophoretic bands barely moved in gel analysis.These results indicated that circular DNA was successfully synthesized and can trigger rolling circle amplification reaction.3.Optimization of experimental conditions:high affinity of aptamer-primer（APP2）was selected.The optimal concentrations of protein G,capture antibody and aptamer-primer were confirmed at 15μg·mL^-1,20μg·mL^-11 and 200 nmol·L^-1,respectively.The optimal reaction time of rolling circle amplification was 60 min,and the suitable pH of working solution was obtained at pH 4.0.4.Analytical performance evaluation of the biosensor:the repeatability of the biosensor was studied by measuring different concentrations(1.0×10² fg·mL^-1,1.0×10⁴ fg·mL^-1,1.0×10⁶ fg·mL^-1)of MPT64 for 10 times.The relative standard deviation（RSD）were 3.66%,3.76%,and 2.60%,respectively（n=10）.The method had good specificity,it responded to MPT64 only.The biosensor had good linear relationship at the range of 5.0 fg·mL^-11 to 1.0×10⁶ fg·mL^-1.The detection limit was 0.34fg·mL^-1.The retained current signal was 97.31%,95.21%,and 90.23%,respectively,after storing in 4℃at 5,10,and 15 day.5.Recovery test:the serum samples were spiked with MPT64.And the final concentrations were 10.0 fg·mL^-1,1.0×10³ fg·mL^-1and 1.0×10⁵fg·mL^-1,respectively.The recovery was 97.97%,106.5%,and 98.95%,respectively.ConclusionIn the present work,a novel biosensor was constructed based on the synergetic signal amplification strategy of GO@Fe₃O₄@Pt nanocomposite and rolling circle amplification.It had a wide linear range,high sensitivity,good specificity,and can be used to detect MPT64 in blood samples.Moreover,the detection process can be completed within 4 h which might provide forceful laboratorial support for the early diagnosis of tuberculosis.