Use of Gb3 trisaccharides with oligo-EG linker for the detection of Escherichia coli O157:H7 Shiga toxins present in a fresh vegetable salad

Two kinds of globotriaosyl (Gb₃) trisaccharides (1 and 2) with an azido (-N = N = N) group at the end of an aglycon oligoethylene glycol group (EG₃ for 1 and EG₁₂ for 2) were prepared. Each of the Gb₃ products was immobilized on a silicon nitride (SiN) chip with a Huisgen coupling reaction. The derived Gb₃/SiN chips were applied as sensor chips in a microfluidic flow system using reflectometric interference spectroscopy (RIfS). When examined with Escherichia coli O157 Shiga toxin (Stx1) as the target toxin and bovine serum albumin (BSA) as the negative control, the microfluidic RIfS analysis has indicated that Gb₃/SiN chips with the elongated EG₁₂ group can suppress a non-selective adhesion of BSA more effectively than those with an EG₃ linker. A spike and recovery test using a Japanese cucumber salad has indicated that chromophoric substances contained in foods interfere with the direct RIfS detection of Stxs. This problem was circumvented by a centrifugal filtration process for each of the test samples in advance of the RIfS detection. The pretreated samples make clear RIfS responses to the target Stx1 bound on the surface of the Gb₃/EG₁₂/SiN chip with a detection sensitivity in a 0.1 ~ 0.5 µg/mL range by LOD. One analysis completes within 25 min after the centrifugal filtration process. The microfluidic RIfS detection of Stx1 also applies to test samples taken from an E. coli selective cultivation medium (EC broth), meaning that it will provide a promising tool for determining STEC and other Stxs-producing bacteria that may contaminate various foods other than the Japanese salad.