Stacking-Directed Energy Modulation in Covalent Organic Framework Thin Films for Enhanced Serum Metabolic Profiling of <i>Mycoplasma pneumoniae</i> Infection by Laser Desorption/Ionization Mass Spectrometry

Covalent organic frameworks (COFs) offer a structurally modular platform for designing high-performance laser desorption/ionization mass spectrometry (LDI-MS) substrates. The interlayer stacking configuration of COFs has been identified as a critical parameter governing energy transduction and interfacial charge dynamics, though its influence on LDI-MS performance remains unexplored. Herein, COF with precisely controlled AA/AB stacking configurations was first synthesized by adjusting the catalysts. Compared with AB-stacked COF, the AA-stacked COF with enhanced π-π interaction and ordered stacking exhibited superior photothermal conversion (62.27%), and charge transport, together enabling more efficient desorption/ionization. While implemented as an LDI-MS substrate, the optimized AA-stacked COF nanofilm-assisted LDI-MS platform enhanced 10-fold detection sensitivity of metabolites while maintaining excellent reproducibility (CV < 8%). Furthermore, this platform could be successfully applied to extract serum metabolic fingerprints from children infected with <i>Mycoplasma pneumoniae</i> (MP). A total of 23 differential metabolites were identified, and the multivariate statistical models achieved group separation (AUC = 0.999), which enables clear discrimination between MP patients and healthy controls. This study established a clear structure-function relationship between the interlayer stacking mode of COFs and LDI-MS performance, and provided a robust and reproducible analytical platform for clinical metabolomic analysis.