Microfluidic concentration gradient generators (µ-CGGs) have been utilized to identify optimal drug compositions through antimicrobial susceptibility testing (AST) for the treatment of antimicrobial-resistant (AMR) infections. Conventional µ-CGGs fabricated via photolithography-based micromachining processes, however, are fundamentally limited to two-dimensional fluidic routing, such that only two distinct antimicrobial drugs can be tested at once. This work addresses this limitation by employing Multijet-3D printed microchannel networks capable of fluidic routing in three dimensions to generate symmetric multi-drug concentration gradients. The three-fluid gradient generation characteristics of the fabricated 3D µ-CGG prototype were quantified through both theoretical simulations and experimental validations. Furthermore, the antimicrobial effects of three highly clinically-relevant classes of antibiotic drugs, tetracycline, ciprofloxacin, and amikacin, were evaluated via experimental single-antibiotic minimum inhibitory concentration (MIC), pair-wise and three-way antibiotic combination drug screening (CDS) studies against model antibiotic-resistant Escherichia coli bacteria. As such, this 3D µ-CGG platform has great potential to enable expedited combination AST screening for various biomedical and diagnostic applications.
Keywords: 3D printed, additive manufacturing, microfluidics, concentration gradient generator, minimum inhibitory concentration, combination drug screening, antibiotic, antimicrobial susceptibility testing