Improvement of High Affinity and Selectivity on Biosensors Using Genetically Engineered Phage by Binding Isotherm Screening.

The genetically engineered M13 bacteriophage (M13 phage), developed via directed evolutionary screening process, can improve the sensitivity of sensors because of its selective binding to a target material. Herein, we propose a screening method to develop a selective and sensitive bioreporter for toxic material based on genetically engineered M13 phage. The paraquat (PQ)-binding M13 phage, developed by directed evolution, was used. The binding affinities of the PQ-binding M13 phage to PQ and similar molecules were analyzed using isothermal titration calorimetry (ITC). Based on the isotherms measured by ITC, binding affinities were calculated using the one-site binding model. The binding affinity was 5.161 × 10-7 for PQ, and 3.043 × 10-7 for diquat (DQ). The isotherm and raw ITC data show that the PQ-binding M13 phage does not selectively bind to difenzoquat (DIF). The phage biofilter experiment confirmed the ability of PQ-binding M13 bacteriophage to bind PQ. The surface-enhanced Raman scattering (SERS) platform based on the bioreporter, PQ-binding M13 phage, exhibited 3.7 times the signal intensity as compared with the wild-type-M13-phage-coated platform.