In-situ preparation of norepinephrine-functionalized silver nanoparticles and application for colorimetric detection of tacrolimus in plasma samples

Tacrolimus (Tac) is a well-documented immunosuppressive agent for the prevention of graft-vs-host diseases in several types of organ transplants. The narrow therapeutic window and the individual-variable pharmacokinetics of Tac demonstrate the importance of regular therapeutic drug monitoring (TDM) as an imperative concept for its oral medication regimens. A simple, one-step, selective, and sensitive colorimetric platform is fabricated for the determination of Tac by surface modification of the silver nanoparticles (AgNPs) via norepinephrine (NE) molecules. The attachment of NE and Tac induces the aggregation of the AgNPs, which is observed by color distinction (yellow to brown) and a noteworthy shifting of the absorption peak in the visible region. The fabricated nanoprobe can detect Tac concentrations in plasma samples in two linear ranges from 2 ng/mL to 70 ng/mL and 70 ng/mL to 1000 ng/mL with R2 > 0.99. The limit of detection (LOD) was calculated as low as 0.1 ng/mL. The developed method was applied for the determination of Tac in patient’s plasma samples under Tac medication therapy.


Introduction
Tacrolimus (Tac), also known as FK-506, is a well-documented immunosuppressive agent for the prevention of graft-vs-host disease in the follow-up medication of several types of organ transplants. The immunosuppression mechanism of Tac relates to the formation of a pentameric complex with Ca 2+ , calcineurin, and calmoduline, which inhibits the nuclear factor of activated T-cells (NFAT). NFAT is a necessary factor for the production of interleukin 2 [1][2][3]. The over-dosage administration of Tac leads to some severe consequences and toxicities, like nephrotoxicity. While the under-dosage decreases its efficacy in the prevention of transplant rejection [4][5][6][7]. To overcome this, the whole blood level of Tac must be retained in the range of 5-20 ng/mL [8]. Additionally, the narrow therapeutic window and the individual-variable pharmacokinetics of Tac demonstrate the importance of regular therapeutic drug monitoring (TDM) as an imperative concept for its oral medication regimens [9].
So far, numerous analytical methods have been used for the quantification of Tac, such as liquid chromatography tandem mass spectrometry (LC-MS/MS) [10][11][12][13], ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) [14][15][16], UHPLC [17], enzyme-linked immunosorbent assay (ELISA) [18], enzyme multiplied immunoassay technique (EMIT) [19], automated microparticle enzyme immunoassay (MEIA) [20], electro-chemiluminescence immunoassay (ECLIA) [21], fluorescent aptasensor [22], and electrochemical immunosensor [9]. Some of these methods require extensive training, a high degree of technical ability, a time-consuming process, and high-cost sophisticated equipment, which are the main drawbacks for rapid and real-time routine quantifications [23]. Modern analysis technology appreciates fast, simple training, and sensitive techniques for TDM, which can be operated for on-site applications [9,[24][25][26][27]. The colorimetric sensors have advantages compared with other quick-detection methods such as electrochemical, fluorescent, etc. due to their naked-eye determination, which is more economical without requiring complicated instruments [28]. As an additional feature for simple colorimetric analysis is the possibility of their precise quantifications using smart-phone applications [29,30]. Various types of materials were utilized for colorimetric detection in different media [23,28,31]. For instance, metal nanoparticles (NPs) such as gold (Au), silver (Ag), copper (Cu), and platinum (Pt) recently appealed to global interests as favorable materials for the invention of an appropriate colorimetric nanosensor due to their distinct color through aggregation state in the presence of the target analytes [31][32][33][34][35][36]. Surface Plasmon Resonance (SPR) refers to the phenomenon that the incident light reflects at an angle on the surface of the metal NPs, which causes a decrease in light intensity. As a result, a noteworthy shift in the absorption peak is observed in the visible region (390-750 nm). Today, plasmonic nanosensors offer the high-priority principle for several recent straight-mode color-based detection applications [37,38]. AgNPs are well-known nanoparticles to be inexpensive colorimetric sensors (in comparison to AuNPs) owning to their ease of color visualization, size-shape-dependent features, strong spectral shifts, high SPR properties, biocompatibility, and long-term stability [39,40]. An AgNPs colorimetric assay typically involves the color change from "bright-yellow to red", where the red color expresses the aggregation state [39]. In addition, the surface modification of the AgNPs can effectively enhance the detection performance.
To provide a simple, selective, and sensitive analytical platform and alter the colorimetric sensing ability of AgNPs, norepinephrine (NE) has been utilized for modification of AgNPs to quantify Tac concentrations in plasma samples. NE with two hydroxyl groups as the multifunctional group modifier increases the bonding ability of the AgNPs to the target analyte. Here, the NE is not only used as a reducing agent but also helps stabilize the AgNPs. Both the functionalization of AgNPs with NE and Tac detection occur in a one-step process. The attachment of NE and Tac induced the aggregation of the AgNPs, resulting in a change in the size and absorption peak of the NPs. The developed method was applied for the determination of Tac in patients' plasma samples with Tac oral consumption regimens.

Apparatus
UV-Vis absorption spectra were obtained by Shimadzu UV-2450 UV-visible spectrophotometer (Tokyo, Japan). Zeta potential, dynamic light scattering (DLS, Malvern particle size analyzer, Malvern, UK) and field emission scanning electron microscopy (FESEM) images (FEG-SEM MIRA3 TESCAN, Brno, Czech Republic) were employed to characterize the size and morphology of the prepared AgNPs, respectively. Also, Fourier transforms infrared (FTIR) spectrum was applied by a Shimadzu model FTIR prestige 21 spectrophotometers (Tokyo, Japan).

Preparation of plasma samples
The drug-free plasma, which was already frozen in polypropylene microtubes, was thawed at room temperature before daily use.
The stock solution of Tac (1000 μg/mL) was prepared in ethanol and used for preparation of Tac-spiked plasma samples (100, 10, and 1 μg/mL). In order to precipitate the plasma proteins, both the drug-spiked and blank plasma samples were mixed by ACN with an equal volume of plasma. The samples were vortexed for 1 min to ensure the denaturation procedure and then centrifuged at 6500 rpm for 15 min. The clear supernatant was discarded and utilized for the colorimetric detection process.
Real plasma samples were obtained from patients with Tac consumption regimens who had signed consent forms with the ethical committee approval code (IR.TBZMED.REC.1400.970). These samples were frozen at − 20 • C and melted at room temperature.

General process for tac determination in plasma
A one-step colorimetric method was set for the detection of Tac in plasma samples. To do, Tac-spiked plasma was prepared in different concentrations (0,1,10, and 100 μg/mL) and then the protein precipitation step was performed. Then, about 10 μL of NE (5 mM) and 10 μL of in different concentrations were mixed in a 2 mL microtube and retained for 1 min. In another microtube, a mixture of 50 μL AgNO 3 (10 mM) mixed by 10 μL NaOH (0.1 M) which causes the color of the solution turn to very pale yellow. Then 150 μL phosphate buffer (50 mM, pH 10) and 800 μL deionized water were added. This solution was mixed by the NE-Tac mixture sequentially which leads to color alteration. In presence of Tac the solution color was altered to brown while it is bright yellow in absence of Tac. The obtained solution was vortexed for 30 s and then it was used for UV-Vis spectra records. The same approach was used for blank samples to compare with Tac-spiked results.

Method optimization process
The quantification process may affect by reaction condition such as pH, vortex time, and amount of AgNO 3 , NE and NaOH. The optimization of these parameters is necessary factor to achieve a sensitive and specific analysis method. As the most important factor, pH was adjusted with phosphate buffer which is prepared by dissolving of disodium hydrogen phosphate in deionized water (50 mM). Then, it was adjusted in pH range of 3-11 by NaoH (0.1 M) and HCl (0.1 M). Also, the adequate volume of phosphate buffer was checked due to addition of 150, 250, 500, and 750 μL with final volume of 1000 μL. The amount of AgNO 3 (10 mM) was investigated in five volumes of 20, 40, 50, 75 and 100 μL. The effect of the amounts of NE (5 mM) and NaOH (0.1 M) were tested in five volumes including 10, 20, 30, 40, and 50 μL.

Mechanism of colorimetric detection
It has been confirmed that catecholamines such as dopamine and NE molecules form chemically adherent films on material surfaces through oxidative polymerization under alkaline conditions [41]. Two ortho-located hydroxyl groups of NE transfer two electrons in an oxidative reaction to form a quinone structure on the surface of AgNPs, while the alkylamine groups stabilize the AgNPs [23]. Scheme 1 demonstrates the color and the UV-Vis absorption spectrum alternation in the presence and absence of Tac. Upon addition of Tac, the color of the AgNPs suspension is obviously changed from yellow to reddish brown, proposing aggregation of AgNPs. A main sharp absorption peak of NE-functionalized AgNPs was observed at 410 nm in the absence of Tac, while it turns to a broad peak at 460 nm after the addition of Tac. Due to the Tac-NE binding reaction, AgNPs are aggregated, resulting in a color change. It is noteworthy to mention that no reaction was observed between Tac and AgNPs in the absence of NE and the mixture remained colorless. approximate diameter of 20 nm. This can be related to AgNPs (Fig. 1B). Besides, other particles were demonstrated in the SEM images with an average diameter of 80 nm which confirmed the DLS results as the size of NE-functionalized AgNPs. In addition, Fig. 1D demonstrates the aggregation of the AgNPs after the addition of Tac. The elemental composition of NE-functionalized AgNPs measured by EDX analysis represented the C, N, O, and Ag elemental percentages of 41.73%, 31.17%, 15.68%, and 11.42%, respectively (Fig. S1). The surface charge of the nanoparticles was determined by the zeta potential technique with a value of − 16.8 mV (Fig. S2), which is close to the catecholamine capped nanoparticles [23].

Characterization of NE-functionalized AgNPs
The standard FTIR spectrum of NE demonstrates an overlapped band around 3200-3400 cm -1 which is assigned to the stretch vibration of NH and OH [42]. These peaks are changed to the sharp peak at 3500 cm -1 in the NE-functionalized AgNPs spectrum. This can be related to oxidative reaction of two ortho-located hydroxyl groups of NE and form a quinone structure on the surface of AgNPs [23]. Also, a peak at around 1650 cm -1 indicates the carbonyl group (C=O) of the quinone compounds ( Fig. 2A). Additionally, the FTIR spectrum of Tac shows a sharp peak at approximately 3000 cm -1 , related to the alkenyl C-H stretch, which is eliminated at NE-functionalized AgNPs in the presence of Tac. This shows donor-acceptor binding between the alkene groups of the Tac and quinone structures of NE (Fig. 2B). Fig. 3 shows the absorbance of the NE-functionalized AgNPs in the presence and absence of Tac with the NE and AgNO 3 spectra. The absorption peak of NE-functionalized AgNPs at 410 nm is changed to 460 nm after Tac addition. Furthermore, NE has a broad absorbance peak around 250 nm, which is evident in the NE-functionalized AgNPs spectrum. The absorbance spectrum of AgNO 3 has no significant peak in the UV/Vis range [43].

Effect of pH on the absorbance of the NE-functionalized AgNPs
The influence of pH on the absorbance and wavelength of the NE-functionalized AgNPs was investigated.   agglomerated at acidic pHs (less than 5) upon neutralization of the surface charges, leading to a change in the SPR of the metallic NPs [44]. In addition, it is observed that the effect of pH on maximum wavelength was different at the tested pH range, in which a blue shift is observable from pH 3 to 10.

Effect of various factors on the tac colorimetric determination
The quantification process of Tac by NE-functionalized AgNPs may be affected by reaction conditions such as pH, vortex time, and the amount of AgNO 3 , NE and NaOH. So, these parameters should be optimized to achieve a sensitive and specific analysis method. To check these phenomena, their effect was investigated by the difference in absorbance of NE-functionalized AgNPs (ΔA = A 1 -A 2 ) in the presence (A 2 ) and absence (A 1 ) of Tac (100 ng/mL).
The pH value has an important influence on the absorbance intensity and peak maxima, which were studied in the range of 3-11, with a maximum amount of ΔA around pH 10. In acidic pHs (less than 4), the absorbance intensity and peak of the NE-functionalized AgNPs are changed due to the changes in the surface charge of the nanoparticles. The increasing trend was observed gradually in peak intensity from pH 6 to 10. In addition, the volume of PBS was optimized (150 μL) which is important due to its effect on the regulation of the solution ionic strength (Fig. 4S B).
The amount of AgNO 3 (10 mM) was investigated in five volumes (20,40,50,75, and 100 μL). The amount of ΔA was at its maximum value of 50 μL (Fig. 2S C). The effects of the amounts of NE (5 mM) and NaOH were also tested with an optimized volume around 10 μL where the A 2 was decreased to its lowest value (Fig. 4S D-E). The higher NE concentrations lead to the formation of less monodispersed AgNPs and a low peak intensity of NPs. The adequate duration of the vortex was observed within 1 min for visual detection of Tac (Fig. 4S F).

Calibration curve and validation
According to the general recommendations of the Food and Drug Administration (FDA), measurements for each typical quantitative analytical evaluation of drugs should be validated by four fundamental parameters include determination of (1) calibration curve, (2) accuracy, precision, and recovery, (3) selectivity, and (4) the stability of analyte in spiked samples. All of these parameters were determined for the proposed method using the calibration curve in plasma samples.

Calibration curve
The calibration curve was constructed via the recording of UV-visible spectra of NE-functionalized AgNPs in eight concentrations of the Tac-spiked plasma samples (2,7,50,70,200, 500, 800, and 1000 ng/mL) vis ΔA. According to the guidelines, the recommended therapeutic blood trough for Tac after kidney transplants should range between 5 and 20 ng/mL [45] and higher levels of 20 ng/mL are directly related to side effects such as nephrotoxicity [46]. Thus, the proposed method not only adequately covers the entire therapeutic range of Tac, but also provides an appropriate platform for the evaluation of toxicities and side effects. A comparison between the performance of the developed method and some of the recent analytical approaches for Tac determination is made in Table 1. The proposed platform quantifies Tac in a suitable linear range, which is comparable with chromatography and MS-based determinations [9,11,15,47]. Although chromatographic methods detect lower concentration of Tac in comparison to the developed method, these methods require extensive training, a time-consuming process, and high-cost sophisticated equipment, making them an inappropriate platform for rapid and real-time routine detection. Also, the colorimetric detection process is capable to observed by naked eyes, which can be considered in smartphone design applications. It is noteworthy to mention that the whole blood samples have a complex matrix, which complicates the detection process more than other biofluids like plasma and pharmaceutical forms [22,48,49]. The obtained analytical figures-of-merit of the developed method indicate that the current method has a potential for rapid and sensitive detection in which the process is as simple as being detected by the naked eye, proposing a promising probe for on-site detection applications. There are only a limited number of spectrophotometric methods available for the quantification of tacrolimus. However, our method is more sensitive and versatile than the previously reported methods. We have also validated the capability of our method in real media and successfully detected tacrolimus in patient samples.

Accuracy, precision, and recovery
The precision and accuracy of the analytical method are defined by repeatability (relative standard deviation (RSD%)) and recovery percentage, respectively. The FDA recommends evaluating these values through intra-and inter-day experiments. Three Table 1 The analytical figures-of-merit of the studied approaches for the Tac determination.  concentrations of Tac-spiked samples, including 3 ng/mL (LQC), 30 ng/mL (MQC), and 90 ng/mL (HQC) were prepared and triplicated. The intra-and inter-day precision values were calculated in the range of 2.32-3.2% and 0.97-4.4%, respectively (Table 2). Also, the recoveries were in the range of 88.78%-105.33% with RSDs less than 5%. According to FDA guidelines [56], the evaluated precision and accuracy at each concentration (LQC, MQC, and HQC) should not exceed 15%. So, the obtained results indicated that the developed NE-functionalized AgNPs method had ideal reliability and repeatability for Tac detection in plasma samples.

Selectivity
As a necessity for analytical method validation, the probe selectivity should be investigated to differentiate and determine the intended analyte in the presence of other compounds in the biological samples. The potential interfering substances in a biological matrix may have interacted with probe molecules and altered the results of the analysis.
As Fig. 5 demonstrates, the RE of all the interfering substances is not as significant as the effect of the detection process (less than 10%), which confirms that the developed method successfully determined Tac concentration in real human samples.

Stability
The stability of the proposed probe was checked by the freeze-thaw method. Three aliquots of two low and high concentrations of Tac-spiked samples (7 and 50 ng/mL) were frozen for 24 h, then thawed at room temperature through three cycles, and then applied to the spectrophotometer. Table 3 provides the obtained results of the stability test, which show the performance of the detection process was reduced by less than 20% after three freeze-thaw cycles.

Application to quantify tac in real plasma samples
Four plasma samples were obtained from patients with a Tac consumption regime who had signed consent forms with the ethical committee approval code (IR.TBZMED.REC.1400.970). Table 5 reports the Tac concentration in the patient's plasma samples. The number of male and female patients is equal and the administrated dose of Tac for these patients was between 3 and 8 mg per day. In three patients, Tac concentrations ranged in therapeutic index from 4.23 to 19.36 ng/mL.

Conclusion
In conclusion, a one-step and simple colorimetric platform was developed for Tac detection in plasma samples via NEfunctionalized silver nanoparticles. It was observed that in the presence of Tac, the solution color was altered, the peak intensity decreased and the maximum wavelength was shifted from 410 to 460 nm. The proposed method offers a facile and fast Tac detection assay that is susceptible for on-site detection applications. Tac concentration was quantified in plasma samples with dual linear ranges from 2 ng/mL to 70 ng/mL and 70 ng/mL to 1 μg/mL with R 2 > 0.99. This approach holds some benefits, such as high sensitivity and interference-free recognition, and has been successfully applied for Tac determination in four patients under an oral regime as a recommended approach for routine TDM.

Author contribution statement
Zahra Golsanamlu: Performed the experiments; Wrote the paper. Jafar Soleymani: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper. Afshin Gharekhani: Contributed reagents, materials, analysis tools or data. Abolghasem Jouyban: Analyzed and interpreted the data.

Data availability statement
Data included in article/supp. material/referenced in article.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments
This study was a part of Z.G. PhD project studies and supported financially by Tabriz University of Medical Sciences with registration number of 68609.

Tac
Tacrolimus NFAT Nuclear factor of activated T-cells TDM Therapeutic drug monitoring LC-MS/MS Liquid chromatography tandem mass spectrometry UPLC-MS/MS Ultra-high performance liquid chromatography tandem mass spectrometry ELISA Enzyme-linked immunosorbent assay