Bubble-cell native fluorescence imaging for high-sensitivity label-free protein quantification.

Quantification of low-abundance proteins is critical for biopharmaceutical development, proteomics, and biochemical research; however, existing methods often require trade-offs among sensitivity, sample consumption, and workflow simplicity. Ultraviolet (UV) absorbance at 280 nm remains the most widely used method for routine protein quantification because it is rapid, label-free, and reproducible, but its intrinsic sensitivity limits the analysis of dilute samples, particularly when only microliter sample volumes are available. Here we present a high-sensitivity, label-free method for protein quantification based on native fluorescence imaging (NFI) using a bubble-cell capillary, in which a short, enlarged section is incorporated into an otherwise uniform capillary to enhance fluorescence detection. The enlarged bubble-cell increases the optical interrogation volume, generating a localized fluorescence peak that enables robust peak-based quantification, signal-to-noise analysis, and direct visualization of proteins near the detection limit. Using only 1-2 μL of sample, the method achieved experimentally validated limits of detection of approximately 0.005 mg/mL for bovine serum albumin (BSA) and 0.0025 mg/mL for monoclonal antibodies, representing detection limits approximately one order of magnitude lower than literature-reported values for NanoDrop UV spectrophotometry, while preserving the simplicity of a rapid, label-free, and reagent-free workflow. Method validation demonstrated good reproducibility, satisfactory spike-and-recovery performance, negligible carryover, and reliable quantification over the validated working range. The method was further applied to monitor low-abundance fractions collected during monoclonal antibody charge-variant fractionation, enabling detection of minor protein populations that were not reliably detected using a conventional fluorescence plate reader. The combination of high sensitivity, validated analytical performance, minimal sample consumption, and simple peak-based detection demonstrates the potential of bubble-cell NFI as a practical platform for routine protein quantification and characterization, particularly when sample availability is limited or low-abundance protein species must be analyzed.