Microfabrication of engineered Lactococcus lactis biocarriers with genetically programmed immunorecognition probes for sensitive lateral flow immunoassay of antibiotic in milk and lake water

Micro/nanomaterials display considerable potential for increasing the sensitivity of lateral flow immunoassay (LFIA) by acting as 3D carriers for both antibodies and signals. The key to achieving high detection sensitivity depends on the probe's orientation on the material surface and its multivalent biomolecular interactions with targets. Here, we engineer Lactococcus lactis as the bacterial microcarrier (BMC) for a multivalent immunorecognition probe that was genetically programmed to display multifunctional components including a phage-screened single-chain variable fragment (scFv), an enhanced green fluorescent protein (eGFP), and a C-terminal peptidoglycan-binding domain (AcmA) anchored on BMC through the cell wall peptidoglycan. The innovative design of this biocarrier system, which incorporates a lab-on-a-chip microfluidic device, allows for the rapid and non-destructive self-assembly of the multivalent scFv-eGFP-AcmA@BMC probe, in which the 3D structure of BMC with a large peptidoglycan surface area facilitates the precisely orientated attachment and immobilization of scFv-eGFP-AcmA. This leads to a remarkable fluorescence aggregation amplification effect in LFIA, outperforming a monovalent 2D scFv-eGFP-AcmA probe for florfenicol detection. By designing a portable sensing device, we achieved an exceptionally low detection limit of 0.28 pg/mL and 0.21 pg/mL for florfenicol in lake water and milk sample, respectively. The successful microfabrication of this biocarrier holds potential to inspire innovative biohybrid designs for environment and food safety biosensing applications.