Dihydropteroate synthase based sensor for screening multi-sulfonamides residue and its comparison with broad-specific antibody based immunoassay by molecular modeling analysis

Highlights: • A rapid and highly sensitive biosensor based on DHPS-DHPPP binary complex for the detection of class-SAs was developed. • The sensitivity of the developed biosensor was significantly improved by optimizing some physical and chemical conditions. • The biosensor could detect at least 29 SAs below the MRLs. • The DHPS-DHPPP binary complex was used to detect SAs in milk showing high accuracy and precision. • The difference of recognition mechanism between DHPS and antibody was determined by molecular modeling analysis. -- Abstract: A biosensor that could simultaneously detect multi-analyte as many as in a single test is more favored when facing lots of samples for screening purpose. In such a biosensor, the recognition element with broad specificity and highly affinity is a key reagent for detection spectrum. Here we report a dihydropteroate synthase (DHPS) based biosensor for detecting 29 sulfonamides (SAs) in milk. The biosensor was established in a competitive format where HRP-labeled tracer and free SAs competitively binding to the limited amount of DHPS-DHPPP (6-hydroxymethyl-7,8-dihydropterin diphosphate) binary complex. Compared to other biosensors based on antibodies with the same objective, the current sensor employing DHPS provided not only highly sensitivity but also preponderant uniform affinities for all individual sulfonamide, which has never been achieved before in any immunosensor. However, the difference of recognition mechanism between DHPS and antibody remains unknown. To address this question, we deconstructed the variable region of two monoclonal antibodies produced by our group and then compared the intermolecular forces and key contacting amino acid residues of both DHPS and antibodies to several typical SAs with help of molecular modeling analysis. Results showed the orientation of the ligands in the binding site played a significant role during the recognition with proteins. The optimized assay provided a limit of detection (LOD) of 1.15–14.91 ng mL⁻¹ and dynamic range was ranging from 1.54 to 126.85 ng mL⁻¹ for 29 SAs. The developed biosensor finally was validated for five SAs in spiked milk with recovery values ranging from 72.7 to 129.3% and coefficients of variation less than 16.0%. The study showed that the biosensor based on DHPS make it a suitable screening method for the simultaneous determination of total SAs residues in food matrices.