Smartphone-assisted optical quantification of d-penicillamine in human plasm sample using toluidine blue as efficient chromogenic probe: introducing a novel portable device toward therapeutic drug monitoring (TDM).

d-Penicillamine (DPA) is a crucial pharmacological agent widely employed in the clinical management of Wilson's disease, rheumatoid arthritis, and heavy metal poisoning due to its potent metal-chelating properties. Given the narrow therapeutic index of DPA and the critical need for precise monitoring to avoid systemic toxicity, developing rapid, sensitive, and accessible analytical methods is highly demanded by clinicians. This study introduces a novel, cost-effective, and sensitive optical sensor for DPA quantification using Toluidine Blue (TB) as the efficient chromogenic probe. Using smartphone-based Digital Image Colorimetry (DIC) coupled with RGB analysis, DPA was measured in the linear concentration range of 0.25 µM to 10 mM in human plasma samples. A key innovation of this research is the implementation of Parafilm-based micro-pad (PF-µPAD) as a portable, hydrophobic sensing substrate for construction of portable optical sensor, which demonstrated exceptional analytical performance for the chemical interaction of analyte-optical probe (DPA-TB), thereby surpassing the accuracy of conventional vial-based colorimetric method. The underlying molecular interaction mechanism was rigorously validated through Zeta potential measurements revealed a substantial shift from +13.4 to -19.3 mV upon DPA interaction with TB, confirming the formation of stable TB-DPA complexes. Furthermore, the assay exhibited suitable selectivity toward DPA against a broad spectrum of amino acids and biological interferents, with dopamine being the only cross-reactive species observed. Finally, the practical utility of this portable sensing platform in human plasma samples, highlighting its strong potential for clinical diagnostics and Therapeutic Drug Monitoring (TDM).