A Homogeneous Signal-On Strategy for the Detection of <i>rpoB</i> Genes of Mycobacterium tuberculosis Based on Electrochemiluminescent Graphene Oxide and Ferrocene Quenching

Tuberculosis (TB) remains one of the leading causes of morbidity and mortality all over the world and multidrug resistance TB (MDR-TB) pose a serious threat to the TB control and represent an increasing public health problem. In this work, we report a homogeneous signal-on electrochemiluminescence (ECL) DNA sensor for the sensitive and specific detection of <i>rpoB</i> genes of MDR-TB by using ruthenium­(II) complex functionalized graphene oxide (Ru–GO) as suspension sensing interface and ferrocene-labeled ssDNA (Fc–ssDNA) as ECL intensity controller. The ECL of Ru–GO could be effectively quenched by Fc–ssDNA absorbed on the Ru–GO nanosheets. The Ru–GO has good discrimination ability over ssDNA and dsDNA. Mutant ssDNA target responsible for the drug resistant tuberculosis can hybridize with Fc–ssDNA and release Fc–ssDNA from Ru–GO surface, leading to the recovery of ECL. Mutant ssDNA target can be detected in a range from 0.1 to 100 nM with a detection limit of 0.04 nM. The proposed protocol is sensitive, specific, simple, time-saving and polymerase chain reaction free without complicated immobilization, separation and washing steps, which creates a simple but valuable tool for facilitating fast and accurate detection of disease related specific sequences or gene mutations

Mechanism of Action of Flavin-Dependent Halogenases

To rationally engineer the substrate scope and selectivity of flavin-dependent halogenases (FDHs), it is essential to first understand the reaction mechanism and substrate interactions in the active site. FDHs have long been known to achieve regioselectivity through an electrophilic aromatic substitution at C7 of the natural substrate Trp, but the precise role of a key active-site Lys residue remains ambiguous. Formation of hypochlorous acid (HOCl) at the cofactor-binding site is achieved by the direct reaction of molecular oxygen and a single chloride ion with reduced FAD and flavin hydroxide, respectively. HOCl is then guided 10 Å into the halogenation active site. Lys79, located in this site, has been proposed to direct HOCl toward Trp C7 through hydrogen bonding or a direct reaction with HOCl to form an −NH2Cl+ intermediate. Here, we present the most likely mechanism for halogenation based on molecular dynamics (MD) simulations and active-site density functional theory “cluster” models of FDH PrnA in complex with its native substrate l-tryptophan, hypochlorous acid, and the FAD cofactor. MD simulations with different protonation states for key active-site residues suggest that Lys79 directs HOCl through hydrogen bonding, which is confirmed by calculations of the reaction profiles for both proposed mechanisms