Genetically Engineered Whole Virus Biorecognition Units for Cu(II) Sensing

A hybrid filamentous fd virus variant (p9V5-p8Cu5) was generated with improved gold (Au) and copper (Cu) binding properties through genetic engineering by displaying five copies of Au-specific V5 peptides (VSGSSPDS) on the head and over 2500 copies of Cu binding Cu5 peptides (HGFANVA) on the elongated body part. Atomic Force Microscopy (AFM) topography imaging revealed the typical filamentous morphology of the engineered p9V5-p8Cu5 phages with a dry-state thickness of around 2-3 nm. Gold nanoparticle (AuNP) binding of the viruses was investigated by Scanning Electron Microscopy (SEM) analyses upon exposure to 20 nm PBS-stabilized AuNPs. The hybrid phages were excessively loaded with AuNPs. Energy Dispersive X-ray (EDX) analyses showed Au, carbon (C), and nitrogen (N) peaks, confirming the presence of AuNPs and the viral organic material. Quartz crystal microbalance (QCM) analyses, performed using Au-coated quartz crystal sensors, gave a net frequency decrease of 250.20 ± 14.37 Hz for the engineered bacteriophages versus 31.40 ± 4.47 Hz for wild-type fd at the 3rd overtone showing their improved Au binding. Enzyme-Linked Immunosorbent Assay (ELISA) analyses conducted with Cu(II)-coated magnetic agarose-based NTA beads revealed the enhanced Cu(II) binding of the hybrid viruses. A colorimetric Cu(II) detection system was validated using 20 nm PBS-stabilized AuNPs and p9V5-p8Cu5 phages. Following the Cu(II) exposure, the AuNP-virus complex solution was treated with 10x PBS to induce AuNP aggregation. The level of AuNP aggregation was correlated with the applied Cu(II) concentration. For the case study, a linear relationship (R² = 0.9926) was obtained as the absorbance ratios at 600 nm and 525 nm (Abs 600/Abs 525) were plotted against the applied Cu(II) concentration from 0 to 15 µM, with an estimated limit of detection (LOD) of around 260 nM (0.0165 mg/L), well below the WHO drinking-water guideline of 1.3 mg/L. No significant interference was observed from Ag(I), Al(III), Au(III), Cd(II), Co(II), Cr(III), Fe(III), Mn(II), Ni(II), Pb(II), Zn(II), Na(I), Ca(II), or Mg(II) at 5 µM. In mixed samples containing Cu(II) together with each competing ion, the response was likewise unaffected, with the exception of Pb(II), which partially suppressed the Cu(II) signal. This study demonstrates the potential of genetically reprogrammed biomaterials as biorecognition units, offering a biocompatible, easily produced, and cost-effective alternative to synthetic peptides in biosensor development.