Bacillus Subtilis Spore And Argonaute System-based Biosensors For Pollutants Detection

The development of biomaterials and biotechnology is important for the fields of environmental remediation,clinic diagnose,and the industrial production.Bacillus subtilis(B.subtilis)spore and Pyrococcus furiosus Ago(PfAgo)-based biosensing was fabricated used for the pollutants detection in this work,as follows:(1)A spore/luminol chemiluminescence(CL)system coupling with glyphosate oxidase was used for glyphosate detection.Firstly,glycine oxidase showed the catalytic activity both of glycine oxidation and weak glyphosate degradation.A glyphosate oxidase B5R18,obtained from Bacillus cereus glycine oxidase through directed evolution,could degraded glyphosate to H2O2.The catalytic efficiency,specificity constant,and affinity enhancement factor of B5R18 towards glyphosate were increased by 2.85×103-fold,2.25×105-fold,and 9.64×104-fold,respectively,compared with those of glycine oxidase from Bacillus cereus.Besides,B.subtilis spore/luminol CL system employed for H2O2 determination.And the limit of detection of H2O2 was 0.050 μmol L-1(S/N=3),and detection range was 0.050-20 μmol L-1(R2=0.99).Based on the above investigation shown,B.subtilis spore-based CL system used for glyphosate detection.and the limit of detection of glyphosate was 0.090 mg L-1(S/N=3),and the detection range was 0.090-75 mg L-1(R2=0.99).The biosensor showed several advantages,such as high-specificity,low-cost,and eco-friendliness,met the demands of practical applications.(2)A spore/3,3’,5,5’-tetramethylbenzidine(TMB)based portable kit used for semi quantitative and quantitative monitoring of F-.The work was investigated awing to the strong inhibition of F-towards CotA-laccase on the surface of spore.Firstly,we developed a simple F-detection kit,with the processes of 2-3 experimental operations,smartphone analysis,realized the high-sensitive detection.and the limit of detection of F-was 0.90μmol L-1(3σ),and the detection range of 5-600 μmol L-1(R2=0.99).Besides,by checking with standard colorimetric card,we could read out the F-range with the semiquantitative mode.The method successfully applied for the F-monitoring in the surface waters and ground water.Finally,the solution-based enzymatic assay and UV-vis spectra used to evaluate the inhibition model of F-on CotA-laccase of spore.And these results implied that F-possibly binds at a site close to the T2 Cu inhibited the oxidation of T2 Cu(I)by O2,further blocking O2 reduction in trinuclear Cu cluster.(3)A spore/2,2’-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid(ABTS)/odianisidine dihydrochloride(o-dianisidine)strategy used for pH monitoring.CotA-laccase showed catalytic activity in the acidic,neutral,and slightly alkaline media.By adding the compound probes of ABTS and o-dianisidine,the colorimetric showed the conversion response to the pH change.Besides,the biosensor realized the pH detection of 1.00-9.00 with the smartphone analysis,and successfully applied in the surface waters,soil,and raw meat samples detection.Finally,we studied the effect mechanism of pH on CotA-laccase of spore for the first.By coupling several testing methods,we obtained that OH-may simultaneously bind at T1 and T2 Cu sites inhibiting the electron transfer between Cu ion sites,which further decreased the catalytic efficiency towards substrates.(4)A PfAgo-mediated Hg2+ detection platform was established.We firstly presented Ago system used for pollutants detection.By the processes of Hg2+ recognition by Aptamer,strand displacement amplification(SDA),the binding and cleavage of PfAgo,and the fluorescence signals readout,we realized the high-specificity detection of Hg2+.