An Integrated Digital Microfluidic Platform with Titanium Dioxide Nanoparticles-Assisted Laser Desorption Ionization Mass Spectrometry for Microbial Analysis

Digital microfluidics (DMF) offers flexible manipulation of discrete droplets, enabling efficient sample processing and integration with various analytical techniques such as mass spectrometry (MS). While recent efforts have been dedicated to coupling DMF with electrospray ionization MS, the integration of DMF with laser desorption ionization (LDI) MS remains unresolved, yet it is crucial for microbial fingerprinting and metabolic analysis. In this study, we introduce an integrated DMF-LDI-MS system for rapid and automated microbial metabolic analysis. By modifying the DMF chip's top substrate with a hydrophilic spot for sample collection and incorporating an adaptor LDI plate, samples processed on the DMF chip can be directly analyzed using LDI-MS. The addition of titanium dioxide nanoparticles during sample processing on the DMF chip effectively eliminates the matrix interferences, increasing detection efficiency. This system achieves a detection limit of 2.86 × 10^-7 mol/L for verapamil, with a dynamic range of 3 orders of magnitude, comparable to conventional matrix-assisted LDI-MS. We then applied the system to analyze clinically isolated <i>Escherichia coli</i>, facilitating the extraction of metabolites on the DMF chip and comparison of metabolic expressions between clinically isolated Gram-positive and Gram-negative bacteria. Metabolites such as uracil, indoline, and arginine were identified from both bacteria, with alanyl-alanine exhibiting higher levels in <i>E. coli</i> compared to <i>Staphylococcus aureus</i>, indicating its potential as a biomarker for differentiating these clinically important bacterial species. Overall, this integrated DMF-LDI-MS system provides a useful solution for automated microbial analysis and metabolic profiling with minimum sample consumption.