Biosensing Tacrolimus in Human Whole Blood by Using a Drug Receptor Fused to the Emerald Green Fluorescent Protein

Abstract

Tacrolimus (FK506) is an immunosuppressant drug (ISD) used to prevent organ rejection after transplantation that exhibits a narrow therapeutic window and is subject to wide inter- and intra-individual pharmacokinetic fluctuations requiring careful monitoring. The immunosuppressive capacity of FK506 arises from the formation of a complex with immunophilin FKBP1A. This paper describes the use of FKBP1A as an alternative to common antibodies for biosensing purposes. Bioassays use recombinant FKBP1A fused to the emerald green fluorescent protein (FKBP1A-EmGFP). Samples containing the immunosuppressant are incubated with the recombinant protein, and free FKBP1A-EmGFP is captured by magnetic beads functionalized with FK506 to generate a fluorescence signal. Recombinant receptor-drug interaction is evaluated by using a quartz crystal microbalance and nuclear magnetic resonance. The limit of detection (3 ng mL^-1) and dynamic range thus obtained (5-70 ng mL^-1) fulfill therapeutic requirements. The assay is selective for other ISD usually coadministered with FK506 and allows the drug to be determined in human whole blood samples from organ transplant patients with results comparing favorably with those of an external laboratory.

Figure 1

Scheme of the fluororeceptor-based assay for the detection of FK506. (I) The immunosuppressant is recognized by the FKBP1A–EmGFP recombinant protein. (II) Magnetic beads functionalized with FK506 are used to capture free FKBP1A–EmGFP. (III) FKBP1A–EmGFP fluorescence captured by FK506-functionalized beads is monitored following washing.

Figure 2

(A) Main features of the expression vector used to obtain a translational fusion protein consisting of FKBP1A and EmGFP. The vector pQE-T7-2 includes a T7 promotor, the lac repressor (lacI) and lac operator (lacO) to suppress uninduced expression, kanamycin resistance (Kan^R), and a Histidine affinity tag (HisTag). (B) Normalized absorption and fluorescence spectra for EmGFP (green) and FKBP1A-EmGFP (gray) in PBS (10 mM, pH 7.4).

Figure 3

(A) Sensor nanoplatform used to examine FK506 binding to FKBP1A through the HisTag-FKBP1A-EmGFP fusion protein. (B) Normalized frequency shift (black curve) and variation of energy dissipation (red curve) during the adsorption of a 500 nM solution of the HisTag-FKBP1A-EmGFP protein on the dithiobis(C₂NTA-Ni²⁺) layer. (C) Variation of adsorbed protein mass on dithiobis(C₂NTA)-Ni SAM with time. (D) Change in surface mass density as a function of FK506 concentration, with the solid line representing the Langmuir fitting curve.

Figure 4

(A) Chemical structure of FK506. (B) X-ray structure of the FKBP1A–FK506 complex (PDB code 5HUA). (C) STD and reference NMR spectra for a sample containing FKBP1A–EmGFP fusion protein and FK506. The strongest STD signals are highlighted in bold text. Solvent residual signals, labeled with parallel lines, appeared as cancelled STD signals.

Figure 5

(A) Calibration plots for FK506 in phosphate buffer (10 mM, pH 7.4) supplied with 0.05% T20 (black ◆, n = 3) and in extracted whole blood (red ●, n = 3), as obtained by using the recombinant protein FKBP1A–EmGFP and MB-FK506 in PBST. (B) Dose–response plots for FK506 (red ●, n = 3, 8 points), MPA (blue ■, n = 3, 7 points), and Sir (black ◆, n = 3, 8 points) obtained by using FKBP1A–EmGFP as the recognizing element and MB-FK506 in PBST. The results are mean signals ± standard errors of the mean (n = 3).

Figure 6

Comparison of the performance of the proposed fluororeceptor-based assay and the commercial CMIA (RSD ≤ 10%) for quantification of FK506 in whole blood samples from transplant patients.