The aNP-TRAP platform is conceived as a modular, field-deployable system enabling in situ microbial cultivation with simultaneous functional screening of diffusing metabolites. The device comprises three stacked layers and an acrylic base for readout:

i. Cultivation layer: 56 hexagonal wells (17 mm edge-to-edge, 4 mm depth) containing ∼0.75 mL semisolid medium; the top is sealed with a 0.2 µm semipermeable membrane to allow nutrient influx while preventing microbial escape.

Components are held together with lateral bolts and gaskets to ensure structural integrity and sterility; the architecture accommodates sensor materials without compressive stress. While the standard top membrane is 0.2 µm, an optional anisotropic upper film can further restrict outward metabolite escape to enhance signal accumulation. The conceptual architecture and functional layers are illustrated in Figures 1A–C.

Device assembly is performed under sterile conditions, drawing on precedents such as the iChip and diffusion chambers (Nichols et al., 2010; Berdy et al., 2017; Jung et al., 2020). Environmental samples (e.g., soil, rhizosphere, sediments) are suspended in native moisture or sterile buffer (PBS/Ringer). Preprocessing with mild bead agitation and mesh filtration reduces debris while preserving viability. Wells receive low-nutrient semisolid medium (1.5%–2% agar/gellan, cooled to 40°C–45 °C). Inoculation can target limiting dilutions for clonal isolation or be performed in bulk for consortia studies.

After inoculation, the bottom of the cultivation layer is sealed with the gradient-porosity membrane and the top with a 0.2 µm membrane. Compression sealing with gaskets and fasteners yields a robust, portable unit. The device can be embedded 5–10 cm in the target matrix or suspended in water using tethers, enabling passive nutrient/signal exchange under near-native conditions. Alternative pore sizes can accommodate fungi/yeasts (Berdy et al., 2017). A lateral schematic of mass transfer and sensing modalities is provided in Figure 2.

The detection layer contains biosensors immobilized in paper/hydrogel/agarose matrices aligned to the wells. Three modalities are envisioned:

Antibacterial: Escherichia coli JW5503-1 + resazurin in hydrogel (PVA or low-melting agarose). Inhibitory activity suppresses metabolic reduction (blue → pink), generating a retained-blue signal (Allen et al., 2022; Sarker et al., 2007).

Antifungal: Candida albicans embedded in redox-sensitive hydrogel with resazurin. Depending on screening goals, alternative fungal sensors (e.g., Saccharomyces cerevisiae, Aspergillus nidulans) may be used.

Quorum-sensing inhibition: Chromobacterium violaceum CV026 in hydrogel with 10–20 µM C6-HSL as inducer; inhibitors suppress violacein pigmentation (McClean et al., 1997; Duddy and Bassler, 2021; Miller and Bassler, 2001). If CV026 stability is suboptimal, wild-type C. violaceum is a viable alternative. The layered configuration and mass flow are depicted in Figure 2.

Viability/specificity in mixed settings: No experiments were performed; feasibility and specificity remain hypothetical and are extrapolated from prior biosensor uses under controlled conditions.

In situ incubation is expected for 3–10 days under ambient conditions (shorter windows help dye/signal stability (Demir et al., 2024)). Monitoring can be visual or via portable imaging; simple image analysis (e.g., ImageJ) can extract RGB/hue measures. Internal controls include uninoculated wells and calibration wells with known compound concentrations. If weak signals are observed, devices may be redeployed to allow signal maturation and microbial proliferation. Thresholds and quantification steps follow standard colorimetric workflows and are specified in Table 1 (Imaging and quantification).

Wells exhibiting reproducible biosensor responses (e.g., retained blue, pigment loss) are prioritized for isolation and scale-up. Downstream characterization includes MALDI-TOF MS, LC-MS/MS, and 16S/ITS sequencing for dereplication and producer prioritization (Ling et al., 2015). Each well’s position is traceable via barcoding or a grid map, and active fractions are dereplicated to prioritize novel candidates. For early validation, we recommend using antimicrobial standards (e.g., ampicillin, nystatin) and AHL analogs to calibrate sensor response functions.

Clarification on “simulations”: In this manuscript, “simulations” refers to conceptual, order-of-magnitude estimates and internal consistency checks (e.g., dimensional analysis, spreadsheet-level parametric sweeps) derived from literature parameters—not to the execution of numerical solvers (no FEM/CFD, COMSOL®, or MATLAB® runs were performed). Reported values are illustrative and for design guidance only.

To evaluate theoretical feasibility, estimates were structured following the logic typical of COMSOL®/MATLAB® model setups (again, not executed):

Nutrient diffusion: A 0.2 µm polyethersulfone (PES) membrane (∼100 µm thick) with D ≈ 5–7 × 10^-6 cm² s^-1 supports equilibration within ∼2–6 h for common nutrients (Stewart, 2003).

Directional transfer: Anisotropic transport (forward D = 5 × 10⁻⁷; reverse D = 5 × 10⁻⁹ cm² s^-1) accumulates metabolites (e.g., violacein, rifamycin) in the detection zone within ∼6–10 h, with >95% reflux suppression (Zhao et al., 2022; Hou et al., 2019).

Containment: Hydrophobic antibiotics (logP >1; MW > 400 Da) show >98% retention at 24 h; smaller/hydrophilic molecules exhibit polarity/size-dependent back-diffusion.

Sensor kinetics (illustrative): antibacterial—>50% viability-proxy drop in 4–6 h at ≥10 μg mL^-1; antifungal—>80% metabolic signal decline within ∼8 h at ∼25 μg mL^-1; QS inhibition—∼70% violacein repression within ∼10 h for IC₅₀ ≈ 5–20 μg mL^-1.

Table 2 Key modeling assumptions and parameter ranges (membrane pore size/thickness; diffusion coefficients; anisotropy targets; retention criteria by logP/MW; inducer dosing for CV026; time-to-signal windows; field incubation ranges), compiled for transparency and to guide early prototyping.