Microfluidic Focusing of Heterogeneous Bacterial Immunocomplexes for Reproducible SERS Immunoassays

Magnetic bead-based surface-enhanced Raman scattering (SERS) immunoassays enable sensitive and multiplexed bacterial detection. However, bacterial targets form heterogeneous magnetic bead-bacterium-SERS nanotag immunocomplexes that vary in size, morphology, and nanotag loading. In static capillary-based measurements, different numbers of heterogeneous immunocomplexes are therefore located within the laser focal volume at each measurement position, resulting in large signal fluctuations and poor measurement reproducibility. In this study, we developed an elasto-inertial microfluidic platform that enables three-dimensional focusing of bacterial immunocomplexes for reproducible quantitative SERS measurements. Bacterial immunocomplexes suspended in 0.5% (w/v) poly(ethylene oxide) were focused near the center of a straight microchannel and continuously passed through a fixed Raman measurement region. High-speed imaging and cross-sectional Raman mapping confirmed central focusing of the immunocomplexes in both the lateral and vertical directions. Compared with static capillary-based measurements, the coefficient of variation decreased from 41.17% to 7.85% for Staphylococcus aureus and from 64.62% to 6.42% for Pseudomonas aeruginosa. The corresponding R2 values increased from 0.857 to 0.985 and from 0.680 to 0.971, respectively. The corresponding limits of detection were 2.32 × 104 CFU/mL for Staphylococcus aureus and 8.50 × 104 CFU/mL for Pseudomonas aeruginosa. The same focusing strategy was further applied to simultaneous detection of the two bacterial targets using spectrally distinguishable Raman reporters. These results demonstrate that three-dimensional elasto-inertial focusing provides an effective approach for improving the reproducibility of quantitative single- and multiplex bacterial SERS immunoassays.