Biosensing Tacrolimus in Human Whole Blood by Using a Drug
Receptor Fused to the Emerald Green Fluorescent Protein
Bettina Glahn-Martínez, Giacomo Lucchesi, Fernando Pradanas-González, Ana Isabel Manzano,
Ángeles Canales, Gabriella Caminati, Elena Benito-Peña,* and María C. Moreno-Bondi*

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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.

■ INTRODUCTION
Tacrolimus (FK506) is one of the most widely used
immunosuppressant drugs (ISDs) against organ rejection
after transplantation. This compound is a hydrophobic
macrolide antibiotic possessing a 23-membered lactone ring
(Figure S1) and typically isolated from the bacterium
Streptomyces tsukubaensis.1 FK506 exhibits high inter- and
intra-patient pharmacokinetic variability, which, together with
its narrow therapeutic window, makes therapeutic drug
monitoring absolutely mandatory to adjust and maintain
appropriate doses.2 Maximizing the efficacy of FK506 while
minimizing its toxicity requires its blood concentration levels
in transplant recipients to fall within the range 5−20 ng mL−1.3

Liquid chromatography coupled to tandem mass spectrom-
etry (MS) detection is the currently preferred choice for
determining FK506 in clinical laboratories.4−7 Although this
technique provides accurate, reproducible results, it requires
sizeable investments and skilled personnel. In addition, it
involves long analysis times and time-consuming sample
preparation procedures, which result in increased costs and
make the hyphenated technique unsuitable for a high-
throughput screening. Immunoassays provide a faster, more
economical choice for quantifying FK506. A number of
commercial assays for this purpose are currently available,
including the cloned enzyme donor immunoassay (Thermo

Scientific),8 enzyme-linked immunosorbent assay (ELISA),9

electrochemiluminescence enzyme immunoassays (ECLIA,
Roche Elecsys),10 and enzyme-multiplied immunoassay (Sie-
mens)11 (Table S5). However, immunoassays are also subject
to certain shortcomings such as a limited availability of
antibodies against tacrolimus owing to its toxic character,
antibody cross-reactivity (CR), and variability between the
batches.12 Also, although antibody-based assays have been the
cornerstone for a number of screening methods, they require
using laboratory animals to obtain the antibodies, which is to
be avoided according to the recent recommendations of
European authorities for protecting the animals used for
scientific purposes.13−15 One alternative circumventing the
ethical issues arising from the use of animal-derived antibodies
for bioassay development relies on recombinant proteins with
a high specificity and affinity for a given target. Protein
engineering techniques have considerably increased the batch-
to-batch reproducibility of these biomolecules and enabled

Received: July 19, 2022
Accepted: November 4, 2022

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their cost-effective large-scale production. In humans, FK506 is
recognized by the immunophilin FK506 binding protein 1A
(FKBP1A), with which it forms a complex that inhibits
calcineurin affecting T-cell activation and proliferation.16 Using
FKBP1A�which exhibits good specificity and affinity for the
immunosuppressant�as a selective recognition element can
provide an effective alternative to commonly used antibodies
for the development of FK506 biosensors and bioassays.
FKBP1A has been used both in native form and genetically
modified with a fluorescent protein to examine protein−
protein interactions and cell labeling17−23 and also to evaluate
new ligand substitutes for FK506.24−28 Although FKBP1A has
additionally been used to develop some sensing platforms,29−33

it has never been fused to fluorescent proteins as an alternative
to antibodies for developing FK506 quantification bioassays.
As in antibody-based commercial assays (ELISA, ECLIA), this
approach dispenses with the need for additional reagents to
quantify the binding event.
In this work, we developed a bioluminescent assay to

quantify FK506 in whole blood by using a natural receptor of
the drug genetically labeled with the emerald green fluorescent
protein (EmGFP) as the recognition element. EmGFP was
chosen on the grounds of its good photostability and
fluorescent properties.34 In the assay, the target immunosup-
pressant is recognized by the FKBP1A−EmGFP-fused protein.
After incubation, the magnetic beads functionalized with
FK506 (MB-FK506) are used to capture free FKBP1A−
EmGFP, which emits a fluorescent signal (Figure 1). The
operating conditions of the assay were optimized in terms of
protein concentration, buffer composition, and incubation time
and temperature. The suitability of the proposed assay was
assessed by analyzing whole blood samples from transplant
patients under treatment with the immunosuppressant. The
results were validated by comparison with those of an external
laboratory using a chemiluminescent microparticle immuno-
assay (CMIA). The proposed assay shows promise for the
development of sensitive antibody-free FK506 detection
systems.

■ MATERIALS AND METHODS
Materials. Invitrogen Dynabeads M-270 amine (MB), 2-

(N-morpholino)ethanesulfonic acid (MES), rapamycin (Sir),
Phusion Hot Start II DNA polymerase, chemically competent
E. coli One Shot BL21 Star (DE3) cells, isopropyl β-D-1-
thiogalactopyranoside (IPTG), pRSET-EmGFP plasmid, Ster-
ilin Black Microtiter Plates, and Tween 20 (T20) were
purchased from Thermo Fisher Scientific (Rockford, IL, USA).
DNA primers were supplied by Integrated DNA Technologies
(San Diego, CA, USA). NEBuilder HiFi DNA Assembly
Master Mix and NEB 5-alpha competent E. coli were obtained
from New England BioLabs (Ipswich, MA, USA). N-
Hydroxysulfosuccinimide sodium salt (sulfo-NHS) and N,N′-
diethylcarbodiimide hydrochloride (EDC) were supplied by
Fluorochem (Hadfield, Derbyshire, UK). FKBP1A-
pDONR221 plasmid was purchased from DNASU (Tempe,
AZ, USA) and tacrolimus (FK506) from Sinoway Industrial
(Xiamen, China). Mycophenolic acid (MPA) was from Alfa
Aesar (Karlsruhe, Germany). Kanamycin and phosphate-
buffered saline with 0.05% T20 (PBST, pH 7.4) were
purchased from Sigma-Aldrich (St. Louis, MO, USA).
Dimethyl sulfoxide (DMSO) was from VWR (Radnor,
PA,USA), sodium hydroxide from Scharlau (Badalona,
Spain), bacterial cell lysis buffer from NZYTech (Lisbon,
Portugal), imidazole from Merck (Darmstadt, Germany), and
fluorescein from Acros Organics (Geel, Belgium). QIAprep
Spin Miniprep Kit and pQE-T7-2 vector were supplied by
Qiagen (Hilden, Germany). Finally, HisTrap FF crude
columns and PD-10 columns were purchased from Cytiva
(Chicago, IL, USA).
Ultrapure water obtained from a Millipore Milli-Q water

purification system was used throughout.
Stock solutions of the ISD at a 2 mg mL−1 concentration in

DMSO stored at 4 °C were used to prepare standard solutions
on a daily basis by dilution in PBST (10 mM, 0.05% T20, pH
7.4).
Instrumentation. UV−vis absorption spectra were ac-

quired using a Varian Cary 3-Bio spectrophotometer. Steady-
state fluorescence measurements were made using a Horiba

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.

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Fluoromax-4TCSPC spectrofluorometer equipped with a 150
W xenon lamp.
Emission lifetime measurements were made on an FLS980-

Xd2-T spectrometer from Edinburgh Instruments (Livingston,
UK) equipped with a Horiba 470LH diode laser (463 nm, < 1
ns pulse width) using a 460 nm excitation bandpass
interference filter and a Hamamatsu R928P photomultiplier
that was cooled thermoelectrically at −21 °C. Emission
lifetime values as measured in the multi-channel scaling
mode were extracted from the exponential decay data by
using a nonlinear fitting algorithm in the software FAST
(Edinburgh Instruments, v. 3.5.0). This allowed residuals with
χ2 < 1.2 to be obtained. Emission measurements were made in
optically diluted samples with Amax < 0.1 in an air atmosphere.
A CLARIOstar fluorescence reader from BMG Labtech

(Ortenberg, Germany) was used for microplate measurements.
The instrument was operated, and the data was processed by
using MARS, the manufacturer’s original software. The
excitation wavelength was 475 ± 10 nm, and detection was
monitored at 520 ± 10 nm.
Solutions were centrifuged on a miniSpin microcentrifuge

from Eppendorf AG (Hamburg, Germany) and evaporated on
a DNA SpeedVac 110 apparatus from Savant Instruments
(Holbrook,NY, USA), microplates were washed in a Hydro-
Flex plate washer from Tecan (Man̈nedorf, Switzerland)
furnished with a magnetic support.
A QCM-Z500 quartz crystal microbalance (QCM) with

impedance monitoring from KSV Instruments Ltd. (Helsinki,
Finland) equipped with a thermoelectric module from Oven
Instruments (Mechanicsburg, PA, USA) was used for the
analysis of the FK506-protein interaction. The resonant
frequency shift (Δf) and change in energy dissipation (ΔD)
of an Au-coated AT-cut 5 MHz sensor from Nordtest srl
(Serravalle Scrivia, AL, Italy) were recorded at the resonance
frequency ( f 0) and its 3rd, 5th, 7th, 9th, and 11th overtones.
The active area of the sensor was 0.785 cm2. The temperature
was kept constant at 20.0 ± 0.1 °C by using a Peltier element
connected to the thermoelectric module. Changes in resonance
frequency (Δf) and energy dissipation (ΔD) were monitored
through multiple odd overtones with a fundamental frequency
of 5 MHz.
Fusion Protein Characterization by NMR Spectrosco-

py. Saturation transfer difference (STD) curves were acquired
from 2048 scans performed in D2O phosphate-buffered saline
(PBS) buffer at 298 K on a Bruker 700 MHz spectrometer
equipped with a cryoprobe. The sample concentration used
was 0.3 mM for FK506 and 6 nM for the FKBP1A−EmGFP
protein (ligand/protein ratio 50:1). STD at the resonance
frequency was set to −0.3 ppm. Two-dimensional TOCSY and
NOESY spectra were additionally acquired from the same
samples for assignment purposes.
Molecular Cloning. For the expression of FKBP1A fused

with EmGFP, the encoding genes were PCR-amplified from
the commercial plasmids by using the Phusion Hot Start II
DNA polymerase and the primer sets stated in Table S1. The
designed primer sets allowed the incorporation of a HisTag
with a TEV site at the N-terminal of FKBP1A and also a
GGGS linker between FKBP1A and EmGFP.
The amplified vector and gene DNA fragments were

combined in a Gibson assembly reaction by using the
NEBuilder HiFi DNA Assembly Master Mix according to the
manufacturer’s instructions. The assembled plasmid (Figure
2A) was transformed into chemically competent DH5α E. coli

cells by heat shocking and plated on LB-agar containing
kanamycin 50 μg mL−1. A single colony was grown overnight
in 5 mL of a LB-kanamycin solution of the same concentration,
and plasmids were extracted and purified by using the QIAprep
Spin Miniprep Kit. The coding sequence of the constructs was
verified to ensure correct assembly by Sanger sequencing
(Figure S2). The plasmid was transformed into chemically
competent BL21 (DE3) E. coli cells by heat shock, and the
resulting cells were plated and selected on LB-agar containing
kanamycin 50 μg mL−1.
Protein Expression and Purification. For FKBP1A−

EmGFP expression, 5 mL of preculture (LB-kanamycin) was
inoculated with a single colony harboring the plasmid and
grown overnight at 37 °C. The preculture was used to
inoculate a main culture of 200 mL (LB-kanamycin) to an
optical density at 600 nm (OD600) of 0.05 and grown at 37 °C
at 175 rpm until an OD600 value of 0.6−0.8 was reached. After
the induction of FKBP1A−EmGFP-fused protein expression
with 100 μL of IPTG (1 M), cultivation was allowed to
continue at the same temperature for 16 h. Then, cells were
collected by centrifugation at 5000g at 4 °C for 10 min and
stored at −80 °C for at least 3 h before resuspension in NZY
bacterial cell lysis buffer (approximately 5 mL g−1 cells)
supplemented with DNaseI (4 μg mL−1), lysozyme (100 μg
mL−1), and protease inhibitor cocktail and lysed by sonication
on ice (VibraCell Ultrasonic Processor 130 W 20 kHz, Ampl
70%) using on/off pulse cycles of 10 s. Cell debris was
removed by centrifugation at 15,000g at 4 °C for 15 min. The
correct expression of the fluorescent protein was confirmed by
examining the fluorescence emission in both soluble and
insoluble fractions and culture media.
The recombinant fluorescent protein was purified from the

cell lysate by the histidine tag (HisTag) present in the N-
terminal using a HisTrap column according to the
manufacturer’s instructions. Briefly, the lysate supernatant
was diluted 1:3 (v/v) with binding buffer (20 mM phosphate,
pH 7.4, containing 500 mM NaCl and 20 mM imidazole) and
loaded onto the HisTrap column at 1 mL min−1. The column
was washed with 30 mL of binding buffer prior to the elution
of the protein with 20 mM phosphate buffer at pH 7.4
containing 500 mM NaCl and 500 mM imidazole. The
presence of the fluorescent protein was monitored by
measuring the fluorescence of 1 mL eluate fractions, and the
fluorescent fractions were pooled.

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 (KanR), 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).

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Imidazole was removed by using a PD-10 column according
to the manufacturer’s instructions, and the protein was stored
in PBS buffer at 4 °C. Finally, protein purity was evaluated by
sodium dodecyl sulfate−polyacrylamide gel electrophoresis
analysis (Figure S3), and concentrations were calculated by
using theoretical extinction coefficients at 280 nm (ExPASy
ProtParam tool).
Magnetic Bead Functionalization. FK506 was immobi-

lized by using hydrophilic Dynabeads M-270 with amine
groups (MB), and carboxylated FK506 (FK506-CO2H)35 was
used in combination with EDC/sulfo-NHS chemistry. For this
purpose, 2 mg of MB were washed three times with 500 μL of
MES buffer (0.1 M, pH 4.7) and resuspended in 1 mL of MES
buffer containing 0.8 μmol FK506-CO2H, 16.8 μmol EDC,
and 35.02 μmol sulfo-NHS. After incubation for 18 h, MB
were washed 3 times with 500 μL of MES and 2 times with
500 μL of PBST. Finally, FK506-functionalized MB were
stored in 400 μL of PBST (5 mg mL−1) at 4 °C.
Fluororeceptor-Based Bioassay. This bioassay was

performed on FK506 standard solutions or spiked samples in
the presence of FKBP−EmGFP and MB-FK506 at the optimal
concentrations. Briefly, fluorescent fusion protein (20 μL, 52.5
μg mL−1) was mixed with 180 μL of either FK506 standard
solution or extracted blood sample in a black 96-well plate and
incubated for 25 min with slow agitation at 18 °C. Then, 10 μL
of functionalized MB were added to the solution, and, after
incubation for 15 min, MB present in the wells were washed
three times with PBST in a HydroFlex plate washer and
resuspended in 50 μL of buffer.
Fluorescence was monitored with a CLARIOstar microplate

reader. Figure 1 depicts the workflow of the assay.
The fluorescence values obtained as the averages of 15

independent well measurements were normalized to the
minimum and maximum signals for logarithmic plotting as a

function of the FK506 concentration. Experimental data were
fitted to the following four-parameter sigmoidal logistic
equation by using the software Origin 2019

=
+

+
[ ]( )

A A
Anormalized signal

1
b

max min

FK506
IC

min

50 (1)

The limit of detection (LOD), which was taken to be the
analyte concentration inhibiting the signal by 10%, and the
dynamic range (DR) to encompass the analyte concentrations
led to a normalized response over the range 20−80%.36

CR was calculated by substituting the optimum assay
parameter values into the following equation

= ×CR(%)
IC

IC
10050

FK506

50
cross reactant

(2)

where IC50 corresponds to 50% binding inhibition.
Analysis of Human Blood Samples. Whole blood

samples were obtained from organ transplant patients and
healthy volunteers at the Clinical University Hospital of
Valladolid, Spain. Samples were collected with permission of
the hospital’s Ethics Committee (no. PI 21-2245) and kept in
EDTA at −20 °C for transport and storage. FK506 was
extracted by using a previously reported method with minor
modifications.37 Briefly, 600 μL of sample was mixed with 600
μL of methanol, sonicated for 5 s three times, and centrifuged
at 12,100g for 30 min. Then, the supernatant was evaporated
to dryness and resuspended in 30 μL of PBS containing 1%
T20 with bath sonication for 5 min. Reconstituted samples
were centrifuged at 12,100g for 5 min, and the supernatant was
diluted with PBS to a final volume of 600 μL. Treated samples
were subjected to the bioluminescence assay and the results

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.

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compared with those obtained by an external laboratory using
the commercial ARCHITECT iSystem from Abbot.

■ RESULTS AND DISCUSSION
Design and Characterization of Recombinant

FKBP1A−EmGFP. EmGFP-tagged FKBP1A was obtained by
cloning the FKBP1A gene in fusion with EmGFP using
standard molecular biology techniques.38 EmGFP and the
FKBP1A receptor in the immunophilin protein EmGFP
construct (Figure 2A) were separated with a glycine−serine
linker (GGGS) to facilitate the FK506 binding. Also, a HisTag
was included at the N-terminus to facilitate protein purification
by affinity chromatography.
PCR-amplified genes were successfully fused in the pQE-T7-

2 vector by using the Gibson assembly reaction, and
FKBP1A−EmGFP was overexpressed as a soluble form in E.
coli BL21 (DE3).
Figure 2B shows the absorption and emission spectra for

FKBP1A−EmGFP in saline phosphate buffer (PBS, 10 mM,
pH 7.4). The absorption and fluorescence peak fell at 484 and
511 nm, respectively. The fluorescence quantum yield,
determined against fluorescein as the standard (Φf = 0.89 ±
0.04 in NaOH),39 was 0.70 ± 0.02 in PBS and the excitation
wavelength λexc = 450 nm. All measurements were made in
triplicate, and absorption at the excitation wavelength was
always below 0.1. The fluorescence lifetime in PBS was 3.07 ±
0.03 ns (Figure S4).
The results for FKBP1A−EmGFP are consistent with the

reported data for EmGFP, which exhibits an absorption peak at
487 nm, an emission peak at 509 nm, and a fluorescent
quantum yield of 0.68 in aqueous solutions.34 The fact that the
shape and position of the fluorescence peaks remained
unchanged indicates that fusion with the FKBP1A protein
did not alter the structure of EmGFP.
Analysis of the FK506−Protein Interaction by QCM.

Binding of FK506 to the recombinant protein FKBP1A−
EmGFP was examined by using a QCM equipped with a
sensor chip that was functionalized with a nanostructured layer
of dithiobis(C2NTA-Ni2+) to provide an immobilization
platform for the fusion protein by HisTag (Figure 3A−C;
see Supporting Information for a detailed description of the
results).
Once the protein layer formed, increasing concentrations of

FK506 from 1 pM to 10 nM were added to the QCM
measuring chamber until a constant frequency shift was
observed. Figure 3D shows variation of the mass surface

density resulting from the frequency shift (Δf 3/3) as a
function of the FK506 concentration. As can be seen, the
analyte was firmly captured by the sensor surface. FK506
seemingly induced no changes in viscoelastic properties in the
protein surface layers. Kinetic parameters were calculated in
accordance with the Langmuir binding model.
The reported solution dissociation constants for FKP1A

binding to FK50640 and Sir41 range from 0.6 to 0.9 nM;
however, the measured FKBP1A−EmGFP binding constant
was extremely small (Kd = 2.1 ± 0.3 pM) as a result of the
protein being immobilized with a restricted conformation�
and of the tests not being comparable with determinations in
solution.
Analysis of the FK506 Interaction with FKBP1A−

EmGFP by NMR Spectroscopy. Characterizing the inter-
action of FK506 with FKBP1A−EmGFP by nuclear magnetic
resonance (NMR) spectroscopy involved acquiring STD
signals. This experiment allows detecting the signals of the
hydrogens that are interacting with the protein; the signals of
the non-interacting hydrogens are cancelled out. Based on the
results (Figure 4), FK506 was effectively recognized by the
FKBP1A−EmGFP fusion protein. Also, the results were
consistent with the structure of the FKBP1A−FK506 complex
as determined by X-ray crystallography (PDB 5HUA);42 in
fact, the strongest STD signals (viz., H2′, CH3 4, and CH3 10)
were due to the ligand protons pointing toward the protein
surface in the structure (Figure 4B).
Assay Optimization. Developing a simple, fast method for

determining FK506 required optimizing the operating
conditions of the bioassay. Different combinations of the
amounts of MB-FK506 (2−8 μg/well) and fluorescent protein
(1.3−6.7 μg mL−1) were used to maximize the sensitivity while
ensuring good precision and low nonspecific binding. As can
be seen from Figure S7, the best results were obtained with 7
μg of MB-FK506 per well and an FKBP1A−EmGFP
concentration of 5.25 μg mL−1. This combination afforded
good sensitivity and a wide DR.
Recognition of FK506 by the recombinant protein was

found to depend strongly on the assay conditions, which
affected the interactions involved. The optimal buffer
composition was identified by using the previous conditions
in the absence (B0) and presence of a 50 ng mL−1

concentration of FK506 (B50). As can be seen from Figure
S8, PBS, HEPES, and TRIS resulted in a highly nonspecific
surface binding of the FKBP1A−EmGFP protein to the
magnetic beads. However, adding 0.05% Tween 20 (T20) to

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.

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the PBS buffer (PBST) minimized nonselective hydrophobic
interactions between MB-FK506 and the fusion protein. PBST
was thus selected for further testing as it provided the best
results in terms of sensitivity (lowest B/B0 ratio) and DR.
The influence of the incubation time on the analytical

response was examined by incubating the FK506 concen-
trations over the range 0−500 ng mL−1 with FKBP1A−
EmGFP for 15, 25, or 35 min before MB-FK506 was added. As
shown in Figure S9, increasing the incubation time from 15 to
25 min increased the sensitivity. Longer times, however,
provided no additional advantage, so a time of 25 min was
chosen for further testing. MB-FK506 stability was assessed by
monitoring a batch over consecutive days. Functionalized
particles remained stable for at least 6 days provided that they
were stored at 4 °C.

Analytical Characterization. Figure 5A shows the
normalized competition curve obtained with FK506 standards
at concentrations from 0 to 500 ng mL−1 in PBST. IC50 was 19
ng mL−1 and the LOD, as calculated at 10% inhibition, 3 ng
mL−1. The DR, taken as 20−80% inhibition,36 spanned
concentrations from 5 to 70 ng mL−1. The average within-
day relative standard deviation (RSD, n = 3) was 11% and the
between-day RSD for assays performed on four non-

consecutive days < 12%. As can be seen from Table S5, the
proposed method is slightly less sensitive than some
commercial immunoassays, whose LOQ range from 0.08 to
0.7 ng mL−1. However, it has a wider DR (Table S5), and its
measuring protocol can be easily automated. Moreover, the
LOD meets the medical specifications for detection and
quantification of the drug in blood; the recommended
therapeutic dose for patients with a normal rejection risk
usually ranges from 5 to 20 ng mL−1.3

Assay selectivity was assessed by determining two ISDs
typically co-administered with FK506 in transplanted patients,
viz., MPA and Sir (Figure S1). As can be seen from Figure 5B,
MPA exhibited negligible CR (<1%). On the other hand, Sir
induced a response similar to that of FK506 (IC50 = 50 ng
mL−1; CR = 38%). Sir, which is structurally similar to FK506,
is also recognized by the receptor in the human body. Thus, it
forms a complex that inhibits the mammalian target of
rapamycin, thereby altering the proliferation of T lymphocytes
and diminishing antibody production as a result.43,44 In any
case, the assay allows either drug to be quantified in transplant
patients.
Evaluation of the Matrix Effect. Matrix effects were

assessed in blood samples subjected to the extraction
procedure described above and spiked with increasing
concentrations of FK506 from 0 to 500 ng mL−1 before the
analysis. Figure 5A compares the dose−response curves
obtained in buffer and pretreated blood. No significant
differences (p > 0.05) were observed, so no dilution was
required to analyze the real samples.
Analysis of Samples. The efficiency of the extraction

procedure was assessed by using the optimized fluororeceptor-
based bioassay to analyze blank whole blood samples spiked at
four different concentration levels (10, 13, 16, and 20 ng
mL−1). Samples were extracted with MeOH under ultrasound,
evaporated, and reconstituted in PBST prior to the analysis. As
can be seen from Table S4, there were no significant
differences between measured FK506 concentrations and
those of immunosuppressant added to the blank samples at
any concentration level.
Finally, the suitability of the proposed bioassay for FK506

quantification in whole blood from transplant patients (Table
S6) was evaluated, and the results were compared with those
obtained by an external laboratory using the commercial
ARCHITECT immunoassay System from Abbott. As can be
seen from Figure 6, there were no significant differences
between the results obtained with the optimized method and
the commercial assay, which testifies to the suitability of the
proposed bioassay. Sample HCUV4 contained an analyte
concentration exceeding 30 ng/mL, so it could not be
quantified with the commercial system. By contrast, the
proposed fluororeceptor-based bioassay allowed it to be
quantified, thanks to its wide DR. No FK506 was detected
in the control samples.

■ CONCLUSIONS
As shown here, recombinant FKBP1A−EmGFP provides an
effective alternative to antibodies for detection and quantifi-
cation of FK506 in human whole blood samples. Recombinant
fusion between FKBP1A and EmGFP avoids the need for
chemical conjugation, thus providing an unlimited production
system that avoids batch-to-batch variability. No matrix effect
was observed in samples treated with methanol, evaporated,
and reconstituted in PBST. Also, up to 96 treated samples can

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).

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be analyzed within 45 min by using a microplate reader. The
optimized fluororeceptor-based assay provides good sensitivity
and was successfully used to analyze the samples from organ
transplant patients treated with FK506. The results compared
favorably with those of a commercial assay typically used by
clinical laboratories to monitor the FK506 concentration.

■ ASSOCIATED CONTENT
*sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.analchem.2c03122.

Chemical structures of the ISDs used in the assays;
details of the primers sets used for PCR amplification;
Sanger sequencing aligment and SDS-PAGE analysis of
FKBP−EmGFP protein, fluorescence decay profile of
the recombinant protein; MALFI-TOF analysis of
FKBP1A−EmGFP; details about the preparation of the
FK506 protein platform on the QCM sensor and data
analysis; optimization of the concentration of the
fluorescent protein and functionalized magnetic beads;
effect of the buffer solution and the incubation time;
comparative summary of analytical methods reported for
the analysis of FK506 in biological samples; and method
validation in whole blood samples (PDF)

■ AUTHOR INFORMATION
Corresponding Authors

Elena Benito-Peña − Department of Analytical Chemistry,
Faculty of Chemistry, Universidad Complutense de Madrid,
28040 Madrid, Spain; orcid.org/0000-0001-5685-5559;
Email: elenabp@ucm.es

María C. Moreno-Bondi − Department of Analytical
Chemistry, Faculty of Chemistry, Universidad Complutense
de Madrid, 28040 Madrid, Spain; orcid.org/0000-0002-
3612-0675; Email: mcmbondi@ucm.es

Authors
Bettina Glahn-Martínez − Department of Analytical
Chemistry, Faculty of Chemistry, Universidad Complutense
de Madrid, 28040 Madrid, Spain; orcid.org/0000-0003-
0128-3113

Giacomo Lucchesi − Department of Chemistry “Ugo Schiff”
and CSGI, University of Florence, 50019 Sesto Fiorentino,
Italy

Fernando Pradanas-González − Department of Analytical
Chemistry, Faculty of Chemistry, Universidad Complutense
de Madrid, 28040 Madrid, Spain

Ana Isabel Manzano − Department of Organic Chemistry,
Faculty of Chemistry, Universidad Complutense de Madrid,
28040 Madrid, Spain

Ángeles Canales − Department of Organic Chemistry, Faculty
of Chemistry, Universidad Complutense de Madrid, 28040
Madrid, Spain; orcid.org/0000-0003-0542-3080

Gabriella Caminati − Department of Chemistry “Ugo Schiff”
and CSGI, University of Florence, 50019 Sesto Fiorentino,
Italy; orcid.org/0000-0002-5947-7757

Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.analchem.2c03122

Author Contributions
All authors have given approval to the final version of the
manuscript
Notes
The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS
This work was funded by the Spanish Ministry of Science and
Innovation (MICINN, RTI2018-096410 B-C21 and PDI2021-
127457OB-C21). B.G.-M. acknowledges Universidad Com-
plutense de Madrid (UCM) for a research grant. All authors
are grateful to Dr. J. Bustamante Munguira of Hospital Clínico
Universitario de Valladolid (Spain) for his invaluable help and
access to the blood samples and also to P. Martínez for helpful
discussions.

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