Control-referenced bacterial bioluminescence reveals a two-axis salivary response phenotype in benign and malignant breast disease.

Saliva is a complex heterogeneous biofluid whose matrix effects can substantially influence biosensor performance. Here, we show that control-referenced bacterial NAD(P)H:FMN oxidoreductase-luciferase kinetics provides a compact functional readout of these matrix effects and reveals a structured salivary response phenotype in breast disease. We analyzed 200 clinically annotated saliva samples representing healthy controls, benign breast disease, early-stage breast cancer and late-stage breast cancer. Three descriptors were extracted from paired sample-control curves: residual luminescence (LI%), fitted area ratio (S) and kinetic shift (b). The area and kinetic descriptors formed a structurally linked S-b coordinate, whereas LI% represented a largely distinct amplitude response. PCA provided an interpretable coordinate representation in which PC1 primarily summarized S-b variation and PC2 was dominated by LI%. This loading geometry was stable to nonlinear age adjustment and repeated equal-size group subsampling. Healthy, benign and malignant groups occupied overlapping but directionally shifted regions of this response space, with benign breast disease showing the largest displacement from healthy controls, primarily along the amplitude-related coordinate. These findings support bacterial bioluminescence curve analysis as a functional approach for interrogating salivary matrix biology and show that curve-derived descriptors retain information not available from endpoint luminescence alone.