Tandem Förster resonance energy transfer induced visual ratiometric fluorescence sensing of tetracyclines based on zeolitic imidazolate framework-8 incorporated with carbon dots and safranine T.

Developing new methods for efficiently detecting tetracycline antibiotics in water has gained much importance. In this work, zeolitic imidazolate framework-8 (ZIF-8) was used as a host to encapsulate carbon dots (CDs) and safranine T (ST) during its self-assembly process to synthesize CDs/ST@ZIF-8, which was then applied as the dual-emissive probe for detecting tetracycline antibiotics. Benefiting from the confinement effects of ZIF-8 and its fluorescence enhancement effects toward tetracycline (TC), a unique tandem Förster resonance energy transfer (FRET) system from CDs to TC and then to ST could be established, with a low limit of detection of 46 nM and excellent selectivity. More importantly, as compared to the CDs/ST system without tandem FRET, the sensitivity of the CDs/ST@ZIF-8 toward TC increased ∼69-fold, and naked eye recognition could also be achieved. Furthermore, by analyzing the R, G, and B values of photos containing different concentrations of tetracycline with the help of a mobile phone and correlating them with the concentration of tetracycline, we can perform the on-site detection of tetracycline, which is convenient, fast, and accurate. This study shows that new insight can be gained for the rational design and application of ratiometric fluorescence sensors based on tandem Förster resonance energy transfer in metal-organic framework materials.

[Ssp DnaB intein-mediated ligation of heavy and light chains of coagulation factor VIII in Escherichia coli].

We studied the ligation of coagulation factor VIII heavy and light chains in Escherichia coli by utilizing the intein-mediated protein trans-splicing. A B-domain deleted factor VIII (BDD-FVIII) gene was broken into two halves of heavy and light chains before Ser1657 which meets the splicing required conserved residue and then fused to 106 and 48 amino acid-containing N-part termed Int-N and C-part termed Int-C coding sequences of split mini Ssp DnaB intein respectively. These two fusion genes were constructed into a prokaryotic expression vector pBV220. Through induction for expression of recombinant protein it displayed an obvious protein band as predicted size of BDD-FVIII protein on SDS-PAGE gel. Western blotting using factor VIII specific antibodies confirmed that this protein band is BDD-FVIII produced by protein trans-splicing. It demonstrated that the heavy and light chains of BDD-FVIII can be efficiently ligated with the Ssp DnaB intein-mediated protein trans-splicing. These results provided evidence for encouraging our ongoing investigation with intein as a means in dual AAV vectors carrying the factor VIII gene to overcome the packaging size limitation of a single AAV vector in hemophilia A gene therapy.