Laser-induced cross-linking GFP-AcmA′ bioprobe for screening Gram-positive bacteria on a biochip

A serviceable bioprobe is one of the important components for the development of microfluidic and lab-on-a-chip systems. In this paper, we report a novel bioprobe, fabricated by laser-induced cross-linking technology, for simple and direct screening of Gram-positive bacteria on a biochip. The AcmA′ protein is known to bind specifically to peptidoglycan (PG), which forms the thick outside layer of Gram-positive bacteria. Moreover, the AcmA′ protein has a much broader spectrum of bacterium types than do antibodies that are more specific to only one bacterium type, because the AcmA′ protein is a generic characteristic of Gram-positive bacteria. Green fluorescent protein (GFP) is generally used as a molecular marker. In this study, GFP was fused with the AcmA′ protein to act as an indicator to trace the AcmA′ binding activity on PG by green fluorescence. The GFP-AcmA′ protein was three-dimensionally structured by laser-induced cross-linking photochemistry technology to fabricate a bioprobe for capturing Gram-positive bacteria. Positive and negative tests on Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus and Streptococcus agalactiae were demonstrated, respectively. Screening is readily performed using optical microscopy observation. The experiments show that only Gram-positive bacteria were bound on the GFP-AcmA′ probes after minutes of incubation and phosphate buffered saline (PBS) rinsing. No binding was observed with the Gram-negative bacteria or with reference probes composed of neutral bovine serum albumin (BSA). Repeated experiments indicate that our bioprobes are reusable. Finally, a 3D wedge-shaped GFP-AcmA′ probe was demonstrated in a microfluidic channel. This study provides a novel platform for convenient Gram-positive bacteria screening that could potentially be used in lab-on-a-chip applications.