Simple and rapid monitoring of doxorubicin using streptavidin-modified microparticle-based time-resolved fluorescence immunoassay

Abstract

Developing a simple analytical method suitable for therapeutic drug monitoring in a clinical setting is key to establishing guidelines on accurate dose administration and the advancement of precision medicine. We devised a simple rapid analytical method through the combination of streptavidin-modified microparticles and a time-resolved fluorescence immunoassay for therapeutic drug monitoring. The analytical performance of this method was investigated and validated using clinical samples. By determination of doxorubicin concentration, the proposed assay has shown a satisfactory linear range of detection (3.8-3000 ng mL^-1) with a limit of detection of 3.8 ng mL^-1 and an IC₅₀ of 903.9 ng mL^-1. The intra and inter-assay coefficients of variation were 4.12-5.72% and 5.48-6.91%, respectively, and the recovery was acceptable. The applicability of the proposed assay was assessed by comparing the determined results with those measured by LC-MS/MS, presenting a satisfactory correlation (R ² = 0.9868). The proposed assay, which shows satisfactory analytical performance, has great potential for application in the field of TDM in the future.

Fig. 1. Schematic illustration of SA-MP-based TRFIA. (A) Labeling of DOX-conjugated ovalbumin complex. (B) Synthesis of SA-MPs. (C) Reaction mode of proposed assay. (D) Structure of doxorubicin. (E) Assay procedure for SA-MP-based TRFIA in detection of DOX.

Fig. 2. Fluorescence characteristics and UV-VIS spectrum of doxorubicin.

Fig. 3. Identification of synthetic antigens of DOX. (A) SDS-PAGE: line 1: blank OVA. Line 2: blank HSA. Line 3–4: DOX–OVA conjugate diluted with PBS at a ratio of 1/10 and 1/5, respectively. Line 5–6: DOX–HSA conjugate diluted with PBS at a ratio of 1/10 and 1/5, respectively. M: standard protein marker. M_w = molecular weight. OVA = 43 kD, HSA = 67 kD. The UV-VIS spectra of three DOX-protein conjugates, including immunogen DOX–KLH conjugate (B), coating antigen DOX–OVA conjugate (C) and DOX–HSA conjugate (D). Blank control: unlinked carrier protein. Positive control: DOX in PBS. DOX-spiked unlinked carrier protein was all diluted in PBS.

Fig. 4. Optimum reaction conditions for SA-MP-based TRFIA in detection of doxorubicin. (A) Working concentration of the SA-MPs. (B) Incubation time. (C) Dilution ratio of biotinylated antibody. (D) Dilution ratio of Eu³⁺-labeled antigen.

Fig. 5. Dilutions and inhibition curve of DOX. (A) Dilution curve of spiked urine samples. (B) Dilution curve of spiked serum samples. (C) Dilution curve of actual clinical serum sample. (D) Inhibition curve of free DOX with the IC₅₀ of 903.9 ng mL⁻¹.

Fig. 6. Dose–response curve of spiked standard samples.

Fig. 7. LC-MS/MS analysis and comparison with SA-MP-based TRFIA. (A) LC-MS/MS analysis of the following DOX standards: 1, 10, 50, 100, 500, and 1000 ng mL⁻¹. Quantitative index: m/z 361.30, fragment of DAU: m/z 321.30. (B) LC-MS/MS calibration curve of DOX. APA = analyte peak area, IPA = IS peak area. (C) Linear correlation between the proposed SA-MP-based TRFIA and LC-MS/MS (R² = 0.9868).