Ascorbic AcidEncapsulated in Apoferritin as ImprovedProtoporphyrin-Sensitized TiO₂ Nanoarrays of a PhotoelectrochemicalNanobody-Based Microfluidic Biosensor for Immunoassay

In this work, a nanobody-based microfluidic photoelectrochemical sensor utilizing protoporphyrin-sensitized TiO₂ nanoarrays as signal transducer was developed for the detection of thymic stromal lymphopoietin (TSLP), a typical biomarker of asthma. The TiO₂ nanoarrays were fabricated on ITO substrates via magnetron sputtering and in situ oxidation, and their visible light absorption and photogenerated carrier properties were improved upon sensitization with protoporphyrin dye. The electron donor ascorbic acid (AA) was encapsulated within apoferritin (ApoFt) and coupled directly to the target TSLP to obtain the ApoFt@AA-TSLP bioconjugate, which was subsequently mixed with different concentrations of TSLP and added to the electrode surface, enabling the quantitative release of AA dependent on the disassembly/reassembly properties of the ferritin shell. The detection process was integrated into a miniaturized microfluidic sensor chip to prevent biomolecular leakage at the sensing interface. Notably, nanobodies were employed in this system instead of traditional monoclonal antibodies to counteract the loss of activity induced by strong alkaline stimulation of the epitope during AA release. The sensor is high specificity, stability, and reproducibility with a sensitive photoelectrochemical response to TSLP in a linear range of 1.00 ng/mL to 1.00 μg/mL and a limit of detection as low as 0.08 ng/mL, demonstrating its significant potential for detecting biomarkers of protein-related diseases.