Detection Of Mycobacterium Tuberculosis CFP10 With Biosensor Based On Nucleic Acid Aptamer

Objective:The recombinant culture filtrate protein 10(CFP10)of Mycobacterium tuberculosis was expressed and purified in vitro,and the CFP10 aptamer was screened and identified.A gold nanoparticle(AuNPs)biosensor based on CFP10 aptamer and a AuNPs mimetic enzyme visual biosensor based on CFP10 aptamer were established to detect CFP10 in serum to assist tuberculosis diagnosis and provide reference for tuberculosis diagnosis.Methods: The recombinant CFP10 was expressed in the strain E Coli BL21 containing recombinant expression plasmid pQE30-cfp10 when induced by IPTG and was purified by metal chelation chromatography.The immunoreactivity of CFP10 was identified by Western blot;BCA method was used to determine the protein concentration of CFP10.The reported high affinity CFP10 aptamer sequence was obtained by queried the literature database and synthesized by the biotechnology company;The binding ability between the aptamer and CFP10 was verified by biotin--avidin-HRP system.Gold nanoparticles(AuNPs)was prepared by sodium citrate heating reduction method.According to the characteristic that nucleic acid aptamer has protective effect on AuNPs in high salt environment,biosensor of AuNPs based on CFP10 nucleic acid aptamer was established to detect CFP10.The peroxidase-like catalytic activity of AuNPs was verified by TMB chromogenic method.The AuNPs were combined with the aptamer which modified with sulfhydryl(-SH)to prepare for AuNPs labeled with aptamer.The CFP10 in serum was captured in the microplate coated with CFP10 polyclonal antibodies and then combined with AuNPs labeled aptamer to form polyclonal antibody-CFP10-AuNPs labeled aptamer complex;Another gold nanoparticles were prepared by trisodium citrate reduction method;The preparation of gold nanoparticles by trisodium citrate reduction requires hydrogen peroxide,and the change of hydrogen peroxide concentration influenced the color of gold nanoparticles;The catalytic ability of AuNPs analog enzyme was regulated by specifically recognizing the target antigen through immune reaction,and the competitive reaction of AuNPs catalytic decomposition of hydrogen peroxide and the reaction between hydrogen peroxide and chloroauric acid to produce gold nanoparticles was also regulated to amplify the detection signal to determine CFP10.As a result,a AuNPs mimetic enzyme visual biosensor for CFP10 detection was constructed.The efficacy of these two methods for diagnosis of tuberculosis was evaluated by detecting the CFP10 in the serum of patients with tuberculosis,healthy persons of physical examination and lung disease without tuberculosis.Results: The recombinant protein CFP10 was successfully expressed and purified.The concentration of CFP10 was 530 μg/ml when measured by BCA method.The CFP10 was shown with good immunoreactivity when identified by Western blot.The sensitivity,specificity,positive predictive value,negative predictive value and diagnostic efficiency of AuNPs biosensor based on CFP10 aptamer were 61.9%,87.8%,83.9%,69.2%and 74.7% respectively.The sensitivity,specificity,positive predictive value,negative predictive value and diagnostic efficiency of AuNPs mimetic enzyme visual biosensor based on CFP10 aptamer was 78.9%,89.7%,90.9%,76.5% and 83.6% respectively.Conclusion:The purified CFP10 was shown with good immunoreactivity;AuNPs prepared by trisodium citrate heating reduction method has good dispersion and peroxidase activity;Detection of CFP10 based on CFP10 aptamer AuNPs biosensor and AuNPs mimetic enzyme visual biosensor have the potential to assist tuberculosis diagnosis.