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DOI: 10.1039/C7RA08724A (Paper) RSC Adv., 2017, 7, 43567-43573

Fast screening of whole blood samples for early detection and monitoring of thyroid diseases

Raluca-Ioana Stefan-van Staden*ab and Grigorina Mitrofana
aFaculty of Applied Chemistry and Materials Science, Politehnica University of Bucharest, 1-7 Polizu St., Bucharest, 011061, Romania. E-mail: ralucavanstaden@gmail.com; Tel: +40751507779
bLaboratory of Electrochemistry and PATLAB Bucharest, National Institute of Research for Electrochemistry and Condensed Matter, 202 Splaiul Independentei St., Bucharest, 060021, Romania

Received 7th August 2017 , Accepted 5th September 2017

First published on 8th September 2017

Abstract

This paper described a fast and reliable method for the screening of whole blood for TSH, L-T4, L-T3 and D-T4 using stochastic sensors. The main advantage of this method is the possibility of tracing the precise diagnosis of thyroid dysfunction from a single whole blood sample by multianalyte screening in one run, with low cost. Six stochastic microsensors based on mixtures between inulins: Frutafruit TEX (TEX) and inulin Inutec (IN), and ionic liquids: L-phenylalanine-tert-butyl-ester-lactate (L-PheC4-Lac), L-alanine-tert-butyl-ester-L-lactate (L-AlaC4-Lac) and L-alanine-tert-butyl-ester-nitrate (L-AlaC4-NO3), physically immobilized in a diamond paste matrix were designed and used for the screening tests. The tests were reliable for the assay of TSH, L-T4, L-T3 and D-T4 in whole blood samples, with recoveries higher than 98.00% with RSD values lower than 1.00% being recorded.

Introduction

The analysis of different biomolecules is of prime importance for life science research and medical diagnostics. Therefore, molecular medicine could represent an important step to personalized medicine, developing also the possibility of early diagnosis.1

Stochastic sensors represent a new class of single-molecule detectors of increasing interest due to their capacity for assessing both qualitatively and quantitatively the analyte of interest, being able to detect simultaneously more than one biomarker.

The stochastic technique consists of measuring the electrical fluctuations of ions that pass through the nanopore at a fixed applied potential. The modulation of the ion current is induced by reversibly binding analytes of interest to the wall of the channel. From the diagrams associated with the individual binding events the signature of the analyte can be identified (the value of toff) and its concentration revealed by ton.

This type of analysis allows the usage of chemical modifiers in order to improve their potential to detect a wide range of molecules.2 Thus in this paper we demonstrated the ability of a mixture made by different types of ionic liquids and inulins to assess four thyroid hormones: thyroid stimulating hormone (TSH), levothyroxine (L-T4), dextrothyroxine (D-T4) and triiodothyronine (L-T3). The enantiorecognition ability of ionic liquids or inulins for the detection of thyroid hormones has been demonstrated elsewhere.3,4

One of the most frequent endocrine diseases are those of thyroid.5 The key assays that are used to detect thyroid dysfunction are serum thyroid stimulating hormone (TSH) and the main circulating thyroid hormones thyroxine (f-L-T4) and triiodothyronine (f-L-T3).1 The level of TSH is used by physicians as a screening test for thyroid diseases. Elevated concentrations of TSH usually represents a sign of a decreased L-T4 or L-T3 production, while suppressed levels can point an excessive activity of thyroid gland.1 The determination of TSH serves not only as a preliminary test for thyroid status, but the high level of TSH is associated with increased thyroid cancer incidence and advanced-stage disease.6

For L-T4 and L-T3 equilibrium dialysis and ultrafiltration represented the gold-standard methods7,8 but they are no longer used, being expensive and time consuming. Other methods such as MS9 LC-MS,10 HPLC,11 RIA12 and CLEIA13 have been developed. Also for the assessment of TSH the most used methods were developed based on immunoassay techniques such as ECLIA,14,15 ELISA,16 IRMA,17 EIA18 and RIA.19 Improved or newer technologies are proving to be viable alternatives, sometimes offering better sensitivity, increased throughput and lower matrix interference. Thus, L-T3, L-T4 and D-T4 have been analyzed with different electrochemical sensors,20–22 biosensors23 and immunosensors.24 For the assay of TSH, immunosensors,25 biosensors26 and optical sensors27 have been reported. Recently, reliable methods of analysis were proposed for assay of biomarkers specific to different illnesses.28–30

This paper proposed a fast and reliable method for the screening of whole blood for TSH, L-T4, L-T3 and D-T4 using stochastic sensors. The main advantage of this method is the possibility of tracing the precise diagnosis of thyroid dysfunction from a single whole blood sample by multianalyte assay in one run, with low cost. Six stochastic microsensors based on mixtures between inulins: Frutafruit TEX (TEX) and inulin Inutec (IN), and ionic liquids: L-phenylalanine-tert-butyl-ester-lactate (L-PheC4-Lac), L-alanine-tert-butyl-ester-L-lactate (L-AlaC4-Lac) and L-alanine-tert-butyl-ester-nitrate (L-AlaC4-NO3) physically immobilized in a diamond paste matrix were designed and used for the screening tests.

Experimental

Materials and methods

Reagents and materials. The standard solutions of free L-T3 (f-L-T3), L-T4 (f-L-T4), D-T4 (f-D-T4) and TSH were prepared in phosphate buffer solution (pH = 7.50). The pH was adjusted using small amounts of 0.1 mol L−1 NaOH or HCl solution. Deionised water obtained from a Millipore Direct-Q 3 System (Molsheim, France) was used for all solutions preparation. Natural diamond powder with particle size of 1 μm (99.9%), free L-T3 (f-L-T3), L-T4 (f-L-T4), D-T4 (f-D-T4), TSH, monosodium, disodium phosphate and ionic liquids L-phenylalanine-tert-butyl-ester-lactate (L-PheC4-Lac), L-alanine-tert-butyl-ester-L-lactate (L-AlaC4-Lac) and L-alanine-tert-butyl-ester-nitrate (L-AlaC4-NO3) were purchased from Sigma Aldrich (Milwaukee, USA). Inulin Frutafruit TEX (TEX) was provided by Sensus (Roosendaal, Netherlands) and inulin Inutec (IN) was provided by Orafti Non Food (Oreye, Belgium). Paraffin oil was purchased from Fluka (Buchs, Switzerland).

Standard solutions of different concentration were obtained by serial dilution. All solutions were fresh prepared before measurements. When not in use, all the solutions were stored in the freezer at 2–8 °C.

Apparatus. PGSTAT 302 was used for the recording of all the chronoamperometric measurements using a software Ecochemie version 4.9. The conventional electrochemical cell was constituted by the working electrode, Ag/AgCl (0.1 mol L−1 KCl) as reference electrode and Pt as counter electrode.
Microsensors design. The matrix selected was modified diamond paste (DP). Ionic liquids such as L-phenylalanine tert-butyl ester lactate (L-PheC4-Lac), L-alanine tert-butyl ester L-lactate (L-AlaC4-Lac), L-alanine tert-butyl ester nitrate (L-AlaC4-NO3) and inulins TEX and IN were selected as modifiers. For the preparation of diamond paste we used natural monocrystalline diamond powder mixed with paraffin oil; 12 μL of each ionic liquid solution (10−3 mol L−1) and 12 μL of each inulin solution (10−3 mol L−1) were added to 100 mg diamond paste to give six modified pastes.

Each modified paste was places in a plastic tube with an internal diameter of the active surface of 300 μm. The electric contact was obtained using an Ag wire inserted into the modified paste. The surface of the microsensor was renewed by polishing with aluminum paper and wetted with deionised water before using. When not in use, the microelectrodes were stored in a dry state at room temperature.

Recommended procedures: stochastic method. All the stochastic measurements were performed using chronoamperometry at a constant potential of 125 mV. The electrochemical cell was filled with blood samples without any pretreatment. The toff values (signatures of thyroid hormones) were identified for TSH, f-L-T4, f-L-T3 and f-D-T4 (Fig. 1). The values of ton were determined and the unknown concentrations of TSH, f-L-T3, f-L-T4 and f-D-T4 were obtained by inserting the value 1/ton in the related equation of calibration (1/ton = f (conc)).
image file: c7ra08724a-f1.tif
Fig. 1 Screening the whole blood for f-L-T3, f-L-T4, f-D-T4 and TSH using the microsensor based on: (a) IN-L-Ala-C4-NO3; (b) IN-L-Ala-L-Lac; (c) IN-L-Phe-C4-L-Lac; (d) TEX-L-Ala-C4-NO3; (e) TEX-L-Ala-L-Lac; (f) TEX-L-Phe-C4-L-LAC.
Sample preparation.
Whole blood samples. Whole blood samples were obtained from 9 patients, who have been previously evaluated in the Elias Emergency University Hospital Bucharest. All experiments were performed in compliance with the EU guidelines, and approved by the medical ethics committee at University of Medicine and Pharmacy “Carol Davila”, Bucharest, Romania (Ethics committee approval no. 11/2013). Informed consents were obtained from human participants of this study. The apparatus cell was filled with the whole blood samples and the stochastic measurements were performed. The unknown concentrations were determined from the calibration graphs as described above in the Recommended procedure section.

Results and discussions

Response characteristics of the stochastic sensors

The response of stochastic sensors is based on channel conductivity, when a fixed potential is applied. Accordingly, for the assay of the thyroid hormones a potential of 125 mV vs. Ag/AgCl was applied. The measurement process of a single molecule is taking place in two stages: the first stage on which the molecule is entering the channel, blocking it – when the current is dropping to zero value (the time designed as toff, that the molecule needs to go into the channel is called signature of the analyte and depends on the size and geometry of the molecule, its capacity of unfolding and of velocity needed to enter the channel); the second stage is the one on which binding with the wall of the channel are taking place (the time spend for these processes is called ton – which is measured between two toff events):3,4
Ch(i) + f-L-T3(i) ⇔ Ch·f-L-T3(i)

Ch(i) + f-L-T4(i) ⇔ Ch·f-L-T4(i)

Ch(i) + f-D-T4(i) ⇔ Ch·f-D-T4(i)

Ch(i) + TSH(i) ⇔ Ch·TSH(i)
where Ch is the channel, and i is the interface, followed by redox processes. In the case of simultaneous investigation of multianalytes, each of them entering the channel in an order given by their size, geometry, stereochemistry, capacity of unfolding and velocity of passing through the opening of the channel; therefor for each of them their signature (toff value, Fig. 1) followed a peak are seen in the diagram obtained after screening of whole blood.31–33

Response characteristics are shown in Table 1. Different signatures of TSH, f-L-T4, f-L-T3 and f-D-T4 were obtained for the proposed sensors; there are only two sensors for which the differences between the signatures of TSH, f-L-T4, f-L-T3 and f-D-T4 are very small: the sensors based on IN-L-Ala-NO3, and TEX-L-Phe-C4-L-Lac and therefore difficult to assess in real samples. All sensors exhibited high sensitivity for the simultaneous assay of TSH, f-L-T4, f-L-T3 and f-D-T4. The lowest limit of determination for f-L-T3 was achieved using the sensor based on IN-L-Ala-C4-L-Lac, while for f-L-T4 was achieved using the sensor based on IN-L-Phe-C4-L-Lac, and for f-D-T4 the lowest limit of determination was given by the sensor based on TEX-L-Ala-C4-L-Lac. For TSH assay, no differences in limit of determination were recorded when the six sensors' response characteristics were determined. Regarding all response characteristics as well as the signatures of the hormones, the sensor of choice for simultaneous assay of thyroid hormones is the one based on TEX-L-Ala-C4-NO3-L-Lac.

Table 1 Response characteristics of the stochastic microsensors based on inulins and ionic liquids stochastic for the assay of f-L-T4, f-D-T4, f-L-T3 and TSH
Microsensors based on: Signature of the enantiomer (toff) Sensibility Linear concentration range Quantification limit: Equation of calibration; correlation coefficient
f-L-T3 (mol L−1 s−1)
IN-L-Ala-C4-L-Lac 1 3.08 × 1010 4 × 10−13 to 10−12 4 × 10−13 1/ton = 0.04 + 3.08 × 1010 × C; r = 0.9967
IN-L-Phe-C4-L-Lac 1 4.02 × 109 8 × 10−12 to 4 × 10−12 8 × 10−12 1/ton = 0.02 + 4.02 × 109 × C; r = 0.9977
IN-L-Ala-C4-NO3-L-Lac 1 3.00 × 1010 2 × 10−12 to 8 × 10−12 2 × 10−12 1/ton = 0.05+3 × 1010 × C; r = 0.9295
TEX-L-Ala-C4-L-Lac 1.7 2.63 × 109 6 × 10−12 to 10−11 6 × 10−12 1/ton = 0.03 + 2.63 × 109 × C; r = 0.9959
TEX-L-Phe-C4-L-Lac 0.7 7.21 × 1010 10−12 to 4 × 10−13 1 × 10−12 1/ton = 0.01 + 7.21 × 1010 × C; r = 0.9973
TEX-L-Ala-C4-NO3-L-Lac 0.7 2.28 × 107 10−9 to 10−11 1 × 10−9 1/ton = 0.05 + 2.28 × 107 × C; r = 0.0986
[thin space (1/6-em)]
f-L-T4 (mol L−1 s−1)
IN-L-Ala-C4-L-Lac 0.7 5.44 × 108 8 × 10−12 to 10−10 8 × 10−12 1/ton = 0.03 + 5.44 × 108 × C; r = 0.9833
IN-L-Phe-C4-L-Lac 0.7 5.93 × 109 10−12 to 4 × 10−12 1 × 10−12 1/ton = 0.03 + 5.93 × 109 × C; r = 0.9833
IN-L-Ala-C4-NO3-L-Lac 0.7 5.41 × 109 8 × 10−13 to 2 × 10−12 8 × 10−13 1/ton = 0.01 + 5.41 × 109 × C; r = 0.9561
TEX-L-Ala-C4-L-Lac 1.3 5.62 × 106 10−10 to 10−8 1 × 10−10 1/ton = 0.01 + 5.62 × 106 × C; r = 0.9985
TEX-L-Phe-C4-L-Lac 0.5 1.21 × 108 8 × 10−12 to 10−10 8 × 10−12 1/ton = 0.03 + 1.21 × 108 × C; r = 0.9361
TEX-L-Ala-C4-NO3-L-Lac 1.1 5.97 × 108 8 × 10−12 to 10−10 8 × 10−12 1/ton = 0.01 + 5.97 × 108 × C; r = 0.9937
[thin space (1/6-em)]
D-T4 (mol L−1 s−1)
IN-L-Ala-C4-L-Lac 2.1 2.63 × 104 10−6 to 10−8 1 × 10−6 1/ton = 0.03 + 2.63 × 104 × C; r = 0.9758
IN-L-Phe-C4-L-Lac 1.1 2.2 × 109 6 × 10−12 to 10−11 6 × 10−12 1/ton = 0.02 + 2.2 × 109 × C; r = 0.9991
IN-L-Ala-C4-NO3-L-Lac 0.4 3.56 × 109 4 × 10−12 to 8 × 10−12 4 × 10−12 1/ton = 0.02 + 3.56 × 109 × C; r = 0.9543
TEX-L-Ala-C4-L-Lac 2.4 4.8 × 1010 4 × 10−13 to 10−12 4 × 10−13 1/ton = −0.007 + 4.8 × 1010 × C; r = 0.995
TEX-L-Phe-C4-L-Lac 1.0 3.14 × 102 10−6 to 10−4 1 × 10−6 1/ton = 0.04 + 3.14 × 102 × C; r = 0.9411
TEX-L-Ala-C4-NO3-L-Lac 1.8 9.48 × 109 8 × 10−13 to 2 × 10−12 8 × 10−13 1/ton = 0.01 + 9.48 × 109 × C; r = 0.9981
[thin space (1/6-em)]
TSH (UI L−1)
IN-L-Ala-C4-L-Lac 0.4 2.96 × 10−1 5.6 × 10−4 to 5.6 × 10−2 5.6 × 10−4 1/ton = 0.02 + 2.96 × 10−1 × C; r = 0.9994
IN-L-Phe-C4-L-Lac 1.2 5.89 × 10−4 5.6 × 10−1 to 5.6 × 101 5.6 × 10−1 1/ton = 0.03 + 5.89 × 10−4 × C; r = 0.9998
IN-L-Ala-C4-NO3-L-Lac 0.6 3.52 × 103 5.6 × 10−8 to 5.6 × 10−6 5.6 × 10−8 1/ton = 0.03 + 3.52 × 103 × C; r = 0.9994
TEX-L-Ala-C4-L-Lac 0.3 4.34 × 103 5.6 × 10−8 to 5.6 × 10−6 5.6 × 10−8 1/ton = 0.02 + 4.34 × 103 × C; r = 0.9996
TEX-L-Phe-C4-L-Lac 1.2 2.24 × 10−1 5.6 × 10−6 to 5.6 × 10−4 5.6 × 10−6 1/ton = 0.03 + 2.24 × 10−1 × C; r = 0.9993
TEX-L-Ala-C4-NO3-L-Lac 0.4 7.06 × 103 5.6 × 10−8 to 5.6 × 10−6 5.6 × 10−8 1/ton = 0.02 + 7.06 × 103 × C; r = 0.9788

The sensors were used for more than 3 months when their sensitivities' RSD (%) values did not exceed 1.00%, proving that the sensors are stable for this period, when accurate and precise determinations were performed for whole blood samples.

The selectivity of sensors is given for this type of sensors by the signatures recorded for each compound analysed with the same sensor. The sensors were tested for f-L-T3, f-L-T4, f-D-T4, TSH and thyroid hormone receptor; different values were obtained for each of the compounds when the same sensor was used. The different values obtained for the signatures made possible the correct identification of the signal of the hormones in the diagrams (Fig. 1), and accordingly reliable qualitative analysis followed by quantitative analysis.

Analytical applications

The response characteristics, selectivity, and reliability of the microsensors made possible the simultaneous assay of f-L-T3, f-L-T4, f-D-T4, and TSH in whole blood samples. The whole blood samples were analysed as collected from the nine patients, immediately after they were collected from patients. Diagrams were recorded (Fig. 1), and signatures (toff values) identified for f-L-T3, f-L-T4, f-D-T4 and TSH in each diagram. After identifying the signature for each biomarker (f-L-T3, f-L-T4, f-D-T4 and TSH), the corresponding ton value was read (see Fig. 1) and introduced in the equation of calibration of each sensors, to obtain the value of the concentration.

Results presented in Table 2 shown a good correlations between the results obtained for the assay of f-L-T3, f-L-T4, f-D-T4, and TSH in whole blood samples. Recovery tests were performed by comparing the amounts of f-L-T3, f-L-T4, f-D-T4, and TSH determined in whole blood samples using ELISA (the standard method), and the proposed microsensors. The values of recoveries (Table 3) shown for each sensor, and each compound a very good correlation, given by the amount of compound found using the proposed microsensors reported to the amount of the same compound found in the sample blood sample using ELISA, the recovery being expressed as % recovery. All relative standard deviations recorded were less than 1.00%, proving a high precission and reliability of the measurements.

Table 2 Determination of f-L-T4, f-D-T4, f-L-T3 and TSH in whole blood samples using the stochastic microsensors
Sample no. Microsensor based on f-L-T3 (ng dL−1) f-L-T4 (ng dL−1) f-D-T4 (ng dL−1) TSH (μUI mL−1)
1 IN-L-Ala-C4-L-Lac 53.99 ± 0.21 6.99 ± 0.01 0.16 ± 0.01 0.056 ± 0.003
IN-L-Phe-C4-L-Lac 54.05 ± 0.20 6.00 ± 0.05 0.14 ± 0.01 0.061 ± 0.002
IN-L-Ala-C4-NO3-L-Lac 54.21 ± 0.19 6.81 ± 0.05 0.14 ± 0.02 0.060 ± 0.003
TEX-L-Ala-C4-L-Lac 53.93 ± 0.20 7.08 ± 0.03 0.17 ± 0.02 0.067 ± 0.003
TEX-L-Phe-C4-L-Lac 54.28 ± 0.18 6.06 ± 0.02 0.13 ± 0.03 0.049 ± 0.002
TEX-L-Ala-C4-NO3-L-Lac 54.10 ± 0.10 6.78 ± 0.01 0.19 ± 0.02 0.046 ± 0.002
2 IN-L-Ala-C4-L-Lac 108.06 ± 0.18 1.38 ± 0.05 0.09 ± 0.02 1.440 ± 0.009
IN-L-Phe-C4-L-Lac 107.95 ± 0.18 1.20 ± 0.02 0.06 ± 0.01 1.400 ± 0.004
IN-L-Ala-C4-NO3-L-Lac 107.21 ± 0.21 1.80 ± 0.07 0.10 ± 0.01 1.600 ± 0.005
TEX-L-Ala-C4-L-Lac 107.33 ± 0.19 1.28 ± 0.04 0.12 ± 0.02 1.610 ± 0.004
TEX-L-Phe-C4-L-Lac 108.21 ± 0.20 1.30 ± 0.02 0.13 ± 0.02 1.609 ± 0.004
TEX-L-Ala-C4-NO3-L-Lac 108.08 ± 0.11 1.27 ± 0.02 0.09 ± 0.01 1.590 ± 0.003
3 IN-L-Ala-C4-L-Lac 143.00 ± 0.18 1.000 ± 0.005 0.011 ± 0.003 2.020 ± 0.005
IN-L-Phe-C4-L-Lac 143.02 ± 0.15 1.020 ± 0.007 0.010 ± 0.002 1.920 ± 0.005
IN-L-Ala-C4-NO3-L-Lac 145.60 ± 0.13 0.991 ± 0.003 0.015 ± 0.002 2.190 ± 0.003
TEX-L-Ala-C4-L-Lac 145.69 ± 0.13 0.978 ± 0.005 0.015 ± 0.005 2.000 ± 0.004
TEX-L-Phe-C4-L-Lac 143.78 ± 0.12 0.956 ± 0.006 0.017 ± 0.002 1.940 ± 0.003
TEX-L-Ala-C4-NO3-L-Lac 142.56 ± 0.10 0.966 ± 0.003 0.016 ± 0.001 1.720 ± 0.002
4 IN-L-Ala-C4-L-Lac 11.72 ± 0.17 3.90 ± 0.03 0.12 ± 0.02 0.028 ± 0.005
IN-L-Phe-C4-L-Lac 11.22 ± 0.15 4.10 ± 0.03 0.11 ± 0.02 0.023 ± 0.005
IN-L-Ala-C4-NO3-L-Lac 11.36 ± 0.17 4.66 ± 0.07 0.15 ± 0.01 0.030 ± 0.002
TEX-L-Ala-C4-L-Lac 11.98 ± 0.12 4.25 ± 0.02 0.12 ± 0.01 0.024 ± 0.002
TEX-L-Phe-C4-L-Lac 11.24 ± 0.12 4.52 ± 0.05 0.17 ± 0.03 0.029 ± 0.007
TEX-L-Ala-C4-NO3-L-Lac 12.08 ± 0.11 4.88 ± 0.03 0.11 ± 0.01 0.023 ± 0.001
5 IN-L-Ala-C4-L-Lac 106.21 ± 0.20 1.38 ± 0.02 0.05 ± 0.01 0.330 ± 0.012
IN-L-Phe-C4-L-Lac 104.96 ± 0.18 1.37 ± 0.02 0.05 ± 0.02 0.390 ± 0.012
IN-L-Ala-C4-NO3-L-Lac 106.00 ± 0.18 1.37 ± 0.03 0.06 ± 0.01 0.330 ± 0.015
TEX-L-Ala-C4-L-Lac 105.92 ± 0.22 1.27 ± 0.05 0.06 ± 0.01 0.360 ± 0.013
TEX-L-Phe-C4-L-Lac 105.33 ± 0.15 1.39 ± 0.01 0.07 ± 0.02 0.330 ± 0.017
TEX-L-Ala-C4-NO3-L-Lac 106.02 ± 0.11 1.39 ± 0.03 0.05 ± 0.01 0.308 ± 0.011
6 IN-L-Ala-C4-L-Lac 170.02 ± 0.22 1.08 ± 0.02 0.08 ± 0.01 0.451 ± 0.011
IN-L-Phe-C4-L-Lac 179.21 ± 0.15 1.31 ± 0.04 0.07 ± 0.02 0.414 ± 0.012
IN-L-Ala-C4-NO3-L-Lac 178.24 ± 0.15 1.13 ± 0.03 0.08 ± 0.01 0.482 ± 0.012
TEX-L-Ala-C4-L-Lac 176.66 ± 0.18 1.20 ± 0.02 0.08 ± 0.01 0.445 ± 0.021
TEX-L-Phe-C4-L-Lac 177.22 ± 0.10 1.24 ± 0.03 0.09 ± 0.02 0.443 ± 0.010
TEX-L-Ala-C4-NO3-L-Lac 173.23 ± 0.12 1.27 ± 0.02 0.08 ± 0.01 0.453 ± 0.010
7 IN-L-Ala-C4-L-Lac 163.34 ± 0.18 1.25 ± 0.05 0.09 ± 0.02 1.410 ± 0.008
IN-L-Phe-C4-L-Lac 164.00 ± 0.18 1.22 ± 0.03 0.10 ± 0.01 1.370 ± 0.008
IN-L-Ala-C4-NO3-L-Lac 161.40 ± 0.15 1.34 ± 0.03 0.08 ± 0.01 1.390 ± 0.009
TEX-L-Ala-C4-L-Lac 162.50 ± 0.17 1.21 ± 0.05 0.08 ± 0.01 1.430 ± 0.007
TEX-L-Phe-C4-L-Lac 164.06 ± 0.11 1.30 ± 0.02 0.09 ± 0.02 1.360 ± 0.007
TEX-L-Ala-C4-NO3-L-Lac 164.03 ± 0.12 1.30 ± 0.02 0.10 ± 0.01 1.359 ± 0.005
8 IN-L-Ala-C4-L-Lac 55.48 ± 0.11 0.776 ± 0.009 0.017 ± 0.002 7.200 ± 0.009
IN-L-Phe-C4-L-Lac 55.59 ± 0.12 0.771 ± 0.012 0.015 ± 0.002 7.150 ± 0.009
IN-L-Ala-C4-NO3-L-Lac 55.62 ± 0.12 0.771 ± 0.011 0.014 ± 0.003 7.610 ± 0.011
TEX-L-Ala-C4-L-Lac 55.27 ± 0.13 0.729 ± 0.009 0.017 ± 0.003 7.470 ± 0.012
TEX-L-Phe-C4-L-Lac 55.53 ± 0.11 0.731 ± 0.011 0.015 ± 0.002 7.000 ± 0.009
TEX-L-Ala-C4-NO3-L-Lac 54.09 ± 0.11 0.729 ± 0.013 0.010 ± 0.002 7.240 ± 0.009
9 IN-L-Ala-C4-L-Lac 59.93 ± 0.11 1.12 ± 0.03 0.011 ± 0.003 2.149 ± 0.011
IN-L-Phe-C4-L-Lac 59.98 ± 0.11 1.15 ± 0.02 0.012 ± 0.003 2.180 ± 0.012
IN-L-Ala-C4-NO3-L-Lac 60.60 ± 0.12 1.11 ± 0.01 0.011 ± 0.023 2.200 ± 0.012
TEX-L-Ala-C4-L-Lac 60.67 ± 0.13 1.13 ± 0.01 0.010 ± 0.003 2.198 ± 0.015
TEX-L-Phe-C4-L-Lac 60.22 ± 0.10 1.16 ± 0.02 0.010 ± 0.001 2.230 ± 0.013
TEX-L-Ala-C4-NO3-L-Lac 60.63 ± 0.08 1.17 ± 0.01 0.009 ± 0.001 2.159 ± 0.013

Table 3 Recovery tests of f-L-T4, f-D-T4, f-L-T3 and TSH in whole blood samples
Microsensor based on f-L-T3%, recovery f-L-T4%, recovery f-D-T4%, recovery TSH%, recovery
IN-L-Ala-C4-L-Lac 98.92 ± 0.09 98.70 ± 0.05 99.22 ± 0.04 99.21 ± 0.02
IN-L-Phe-C4-L-Lac 98.72 ± 0.09 98.99 ± 0.05 99.05 ± 0.04 99.20 ± 0.02
IN-L-Ala-C4-NO3-L-Lac 98.90 ± 0.08 98.01 ± 0.06 98.98 ± 0.05 99.43 ± 0.03
TEX-L-Ala-C4-L-Lac 98.50 ± 0.07 98.20 ± 0.07 98.32 ± 0.05 99.32 ± 0.02
TEX-L-Phe-C4-L-Lac 99.01 ± 0.07 99.03 ± 0.07 99.99 ± 0.02 99.99 ± 0.02
TEX-L-Ala-C4-NO3-L-Lac 99.24 ± 0.08 99.56 ± 0.04 99.93 ± 0.04 99.98 ± 0.02

Conclusion

Six stochastic microsensors based on diamond paste modified with inulins and ionic liquids were proposed for the assay of f-L-T4, f-D-T4, f-L-T3 and TSH in whole blood samples. The microsensors were selective, and enantioselective and presented high sensitivity and reliability for the assay of thyroid hormones in whole blood samples. Regarding all response characteristics as well as the signatures of the hormones, the sensor of choice for simultaneous assay of thyroid hormones is the one based on TEX-L-Ala-C4-NO3-L-Lac.

Compared with ELISA and chemiluminescence methods used in clinical laboratories for their determination, the main advantages of the proposed method are: there is no need for sample pretreatment before assay, samples being used as taken from the patient, all four hormones can be determined in one run, low cost, decreased time of determination, high sensitivities and lower limits of determination.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by UEFISCDI PNIII Program PCE Contract nr. 46/2017.

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