A chelating-bond breaking and re-linking technique for rapid re-immobilization of immune micro-sensors

With high sensitivity and specificity to antigen, immune micro-sensors can be used in rapid detection of pathogenic microbial. This study proposes and develops a method for rapidly regeneration of antibody on a resonant micro-cantilever sensor. A nitrilotriacetic acid (NTA) derivative is synthesized with cystine and bromoacetic acid, then added with 2-mercaptoethanol to prepare a mixed self-assembled monolayer (SAM) on Au (111) surface of the cantilever. Ni²⁺ ions are thereafter chelated on the mixed SAM to form a breakable and re-linkable chelating-bond layer. Repeatable cycles of antibody immobilization and erasing are experimentally validated with a detectable marker of synthesized biotinylated poly peptides harboring six histidine residues (named as His-Bio). Two distinguished pathogenic microbial, Escherichia. coli O157:H7 and Bacillus Anthracis, are detected with the rapidly regenerated sensor. The E. coli O157:H7 sensor exhibits a three-time repeated detection to the 10³ CFU/ml concentration microbial. Then, an E. coli O157:H7 sensor is eluted with Tris-HCl (20 mM Tris, 150 mM NaCl, 0.1% Tween 20, pH = 3.0) and rapidly reconstructed into a B. Anthracis sensor by changing the re-immobilized antibody. The cantilever sensor no longer responses to E. coli O157:H7 even in a high concentration of 10⁷ CFU/ml. In contrast, the sensor is experimentally confirmed being resoluble to low concentration B. Anthracis at 10³ spores/ml level. The proposed fast regeneration method is promising in repeatedly or multi-target detection applications of micro/nano immune-sensors, e.g. the resonant micro-cantilevers.