To address the growing pathogenicity and biofilm-mediated drug resistance of Pseudomonas putida (P. putida) in root canal therapy failure and refractory apical periodontitis, and to overcome the bottleneck of "extremely low sample quantity" that hinders the systemic activity evaluation of natural products. This study aims to establish a micro-scale, visual, and high-throughput drug screening platform based on microfluidic chips.

P. putida KT2440 was selected as the model strain, and a green fluorescent protein (GFP) -labeled strain was constructed to establish a microfluidic biofilm model. To accommodate rare natural products, a "qualitative rapid screening" strategy with a fixed threshold of 200 μg/mL was established. Using the length of the fluorescent line segment as a quantitative metric, 40 natural products derived from myxobacteria and plants were blindly screened for anti-biofilm activity. Calcium hydroxide and Ciprofloxacin served as the lower and upper limits of efficacy, respectively, to define the effective screening range. A t-test was used to compare results with the traditional 96-well plate micro-broth dilution method; consistency between the two methods was verified, and sample consumption was compared.

The microfluidic screening platform successfully categorized the 40 compounds into three tiers: Strong, moderate, and ineffective. Ultimately, 6 lead compounds with significant anti-P. putida biofilm activity were identified (positive detection rate of 15%), including novel macrocyclic peptides and certain monoterpenoids. Methodological validation showed high qualitative consistency between the microfluidic chip screening and traditional methods. regarding sample consumption, the platform required only nanoliter (nL) -level volumes per test, with a single compound consumption as low as 8 ng. This represented a sample saving of approximately 15 000-fold compared to the traditional 96-well plate method, significantly reducing the demand for fermentation products.

The microfluidic high-throughput screening platform constructed in this study effectively overcame the technical challenge of sample scarcity in natural product mining through a fixed-concentration qualitative screening strategy. The platform featured extremely low sample consumption, high analytical throughput, and accurate results, providing strong technical support for rapidly locking onto active lead compounds against drug-resistant bacterial biofilms.