(436h) Thyroid hormone selective recognition: Elaboration of levothyroxine-selective molecularly imprinted nanostructured films

Thyroid hormones (TH) are hormones produced in the thyroid gland and play a key role in vertebrate physiology and development, including fetal and post-natal nervous system development and the maintenance of adult brain function [1] . TH family is mainly composed of T3 (3,5,3´-triiodothyronine) and T4 (3,5,3´,5´-tetraiodothyronine) that have similar chemical structure based on tyrosine [2] . In the last years, T4 was the fourth-most prescribed medication in the United States with more than 20 million people buying under medical prescription [3] . Mean levels of T3 and T4 in healthy adults are ranged between 1 and 2 ng/mL, and 50 and 110 ng/mL, respectively [4] . T4 has been proposed as a biomarker for several diseases. While low T4 serum levels have been associated with Beckwith syndrome in infants (below 14.0 ng/mL), hypothyroidism (7.0 – 50.0 ng/mL), and congenital hypothyroidism (8.9 – 57.1 ng/mL), high T4 levels are related to hyperthyroidism (113.0 – 365.0 ng/mL) [5], [6] . A wide range of analytical methods for the detection of TH have been developed, including radioimmunoassay, immunometric assay, chemiluminescence, mass spectroscopy and high-performance liquid chromatography, with low limits of detection.

Molecularly imprinted polymers (MIPs) are synthetic polymers with predetermined selectivity toward a given analyte or a group of structurally related species due to the creation of highly specific sites formed during the synthesis. Hydrogels with submicrometer pores, such as those obtained from colloidal deposits (PC), have a high surface-to-volume ratio that gives them lower resistance to mass transfer. Therefore, the combination of molecular imprinting technology with nanostructured films offers the possibility to develop sensors for rapid detection at low concentrations. In the last 10 years, MIP-PC based sensors have been developed for different compound families such as explosives, pesticides, organophosphates, drugs, antibiotics, hormones among others.

The current work describes the preparation of T4-imprinted polymers by employing a colloidal deposit as a sacrifice template. The obtained materials were characterized, and the recognition capacity, and imprinting efficiency were tested.

Materials and Methods

The colloidal deposits were fabricated by vertical lifting deposition and the films were elaborated using acrylic acid (AA), methacrylic acid (MA) as monomers. The optical properties of the developed materials were characterized by recording their UV-visible reflectance spectra. The swelling capacity of hydrogel films was studied at different pH, their morphological characterization was performed by SEM. The T4 adsorption capacity of AA films elaborated without T4 (AA-NIP) and with a low and high level of impression (AAMIPL and AA-MIPH, respectively) were evaluated through recognition and swelling studies.

Results and Discussion

SEM images of the colloidal deposits and MIPs present a highly periodic ordered structure which could provide a photonic band gap (Fig. 1.a). Obtained films showed a highly interconnected crystalline array (Fig. 1.b). The reflectance peak of the silica colloidal deposits and the obtained nanostructured films was 540 and 409 nm, respectively (Fig. 1.c). The materials showed a pH-sensitive swelling with an increase on water absorption at higher pH values (Fig. 1.d).

An enhancement in the films recognition capacity (RC), with increasing target concentration was observed, leading to a corresponding enhancement in impression efficiency (IE). While films AAMIPL presented an RC value 1.87 times higher than AA-NIP, this parameter increases up to 3.40 for AA-MIP-H, which is related to the higher number of binding sites in the material developed with a greater amount of the target (Table 1). The T4 recognition was also evaluated through a simple gravimetric method. To study the swelling of the MIPs and NIPs after T4 adsorption, samples (approximately 25 mg in cylinder form) were incubated in a 20 ppm target solution for 36 hours, and the swelling degree (SD %) was recorded. Results reported in table 1 demonstrated that MIPs present a higher swelling than NIPs and the SD % also depends on the amount of target present during the imprinting process.

In the current work, it has been demonstrated that AAMIP absorbs T4 by specific interactions, which could change the films reflection spectrum. To evaluate this phenom, plastic-supported AA-MIP-L were incubated in water (0 ppb for 20 min), after that were exposed to a 20 ppb T4 solution and the reflectance spectrum was recorded after each step. Obtained results (Figure 2) showed a peak shift of 16.5 ± 5.0 nm (n=5).

Conclusions

Nanostructured films were developed for the specific adsorption of the thyroid hormone T4. The colloidal deposits elaborated with the vertical lifting methodology showed a narrow size distribution and the obtained colloidal deposits presented a uniformly ordered structure, which provides a template for the elaboration of highly interconnected polymer matrix. The recognition capacity and the imprinting efficiency results showed that the values obtained by the MIPs were higher than the NIPs, demonstrating an efficient molecular imprinting process. Owing to the optical response and the capability of binding T4 hormone, the materials elaborated in the current work have the potential to be applied in the development of fast response photonic sensors for the quantification of T4 in different matrices (blood, for instance).

References

[1] A. C. Schroeder and M. L. Privalsky, “Thyroid hormones, T3 and T4, in the brain,” Front Endocrinol (Lausanne), vol. 5, no. MAR, pp. 1–6, 2014, doi: 10.3389/fendo.2014.00040.

[2] V. F. Samanidou, H. G. Gika, and I. N. Papadoyannis, “Rapid HPLC analysis of thyroid gland hormones tri-iodothyronine (T3) and thyroxine (T4) in human biological fluids after SPE,” J Liq Chromatogr Relat Technol, vol. 23, no. 5, pp. 681–692, 2000, doi: 10.1081/JLC-100101481.

[3] “MEPS summary tables.” https://meps.ahrq.gov/mepstrends/hc_pmed/ (accessed Oct. 25, 2020).

[4] S. Andersen, K. M. Pedersen, N. H. Bruun, and P. Laurberg, “Narrow individual variations in serum T4 and T3 in normal subjects: A clue to the understanding of subclinical thyroid disease,” Journal of Clinical Endocrinology and Metabolism, vol. 87, no. 3, pp. 1068–1072, 2002, doi: 10.1210/jcem.87.3.8165.

[5] B. B. Prasad, M. P. Tiwari, R. Madhuri, and P. S. Sharma, “Enatioselective quantitative separation of d- and l-thyroxine by molecularly imprinted micro-solid phase extraction silver fiber coupled with complementary molecularly imprinted polymer-sensor,” J Chromatogr A, vol. 1217, no. 26, pp. 4255–4266, 2010, doi: 10.1016/j.chroma.2010.04.055.

[6] B. Oerbeck, K. Sundet, B. F. Kase, and S. Heyerdahl, “Congenital hypothyroidism: influence of disease severity and L-thyroxine treatment on intellectual, motor, and school-associated outcomes in young adults,” Pediatrics, vol. 112, no. 4, pp. 923–930, Oct. 2003, doi: 10.1542/PEDS.112.4.923.