Voltammetric sensor based on molecular imprinted polymer for lincomycin determination published by Ural Federal University eISSN 2411-1414 chimicatechnoacta.ru ARTICLE 2023, vol. 10(2), No. 202310210 DOI: 10.15826/chimtech.2023.10.2.10 1 of 8 Voltammetric sensor based on molecular imprinted polymer for lincomycin detection Yulia A. Yarkaeva * , Daria A. Dymova , Marat I. Nazyrov , Liana R. Zagitova , Valery N. Maistrenko Department of Chemistry, Ufa University of Science and Technology, Ufa 450076, Russia * Corresponding author: julijajarkaeva05@gmail.com This paper belongs to a Regular Issue. Abstract For the selective detection of the antibiotic lincomycin, we developed a voltammetric sensor based on a glassy carbon electrode modified with re- duced graphene oxide and polyarylenephthalide containing diphe- nylenethio and diphenyleneoxide fragments in the main chain of the poly- mer in the 1:1 ratio with lincomycin molecular imprints obtained by phase inversion. Using FTIR spectroscopy, electrochemical impedance spectros- copy, cyclic and differential-pulse voltammetry, the electrochemical and analytical characteristics of the sensor were studied. The detection of lin- comycin was carried out by differential pulse voltammetry. The linear con- centration range was 2.5·10–7–5·10–4 M with a limit of detection of 6.8·10– 8 M. It was shown that the presence of molecular imprints increases the sensitivity of the developed sensor in comparisons with a sensor with non- imprinted polymer by a factor of 3.05. Keywords molecularly imprinted polymers polyarylenephthalides voltammetry lincomycin reduced graphene oxide phase inversion Received: 05.04.23 Revised: 26.04.23 Accepted: 26.04.23 Available online: 28.04.23 Key findings ● MIP-sensor for the lincomycin determination based on polyarylenephthalides was obtained by phase inversion. ● The presence of molecular imprints increases the sensitivity of the MIP-sensor by a factor of 3.05. ● MIP-sensor was tested to determine lincomycin in human urine and blood plasma; RSD did not exceed 7.5%, and the recovery was 93–108%. © 2023, the Authors. This article is published in open access under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). 1. Introduction Antibiotics are one of the most important medicines that affect human health. It is difficult to imagine modern med- icine without antibiotics. However, their uncontrolled use has led to antibiotic contamination of ecosystems and food [1–4]. This has increased the already high level of re- sistance of many bacteria to antimicrobial drugs. The con- trol of antibiotic content has become an urgent task for spe- cialists in the field of environmental protection and food quality assessment, analysis of biological objects, and clin- ical medicine [5, 6]. To date, such analytical methods as HPLC [7], capillary electrophoresis [8], FTIR spectroscopy [9], Raman spec- troscopy [10], fluorimetry [11], and microbiological meth- ods [12] are widely used for the detection of antibiotics. Re- cently, electrochemical methods have been used for these purposes, in particular, voltammetry, which makes it pos- sible to quite simply, quickly, and with high sensitivity de- tect drug compounds, including antibiotics, in various ma- trices [13]. Various voltammetric methods, such as cyclic, differential pulse and square wave voltammetry, have been successfully applied with high selectivity and sensitivity for the analysis of drugs and the determination of antibiotics in pharmaceutical dosage forms (tablets, capsules, injec- tions and suspensions) and biological fluids (urine samples, blood and its serum, etc.) [14–17]. When creating sensors for the detection of antibiotics, the main and most com- monly employed approach to modifying electrodes is the use of molecularly imprinted polymers (MIPs) [18]. This al- lows solving the main problem of voltammetry, i.e., insuf- ficient selectivity of detections. Such an approach is analo- gous to antibody-antigen or enzyme-substrate interactions (key-lock interactions) in biological systems. It is based on http://chimicatechnoacta.ru/ https://doi.org/10.15826/chimtech.2023.10.2.10 http://creativecommons.org/licenses/by/4.0/ https://orcid.org/0000-0002-0085-436X https://orcid.org/0000-0001-5602-8609 https://orcid.org/0000-0002-4081-8726 https://orcid.org/0000-0002-7769-7614 https://orcid.org/0000-0001-7831-0269 https://crossmark.crossref.org/dialog/?doi=https://doi.org/10.15826/chimtech.2023.10.2.10&domain=pdf&date_stamp=2023-04-28 Chimica Techno Acta 2023, vol. 10(2), No. 202310210 ARTICLE 2 of 8 DOI: 10.15826/chimtech.2023.10.2.10 the polymerization of a monomer in the presence of a template molecule, with the resulting polymer containing specific cavities of the analyte after its removal from the polymer [19, 20]. Compared to antibodies, MIPs are cheaper, more stable, and withstand a wider range of pH and temperature [21]. The development of MIP for modify- ing electrodes in voltammetry includes several key steps: polymerization or polymer deposition; removal of the tem- plate (usually by a solvent or electrochemical method); in- strumental measurements. MIPs can be obtained using var- ious methods of monomer polymerization in the presence of a template molecule (chemical, electrochemical, photo- polymerization). In addition to polymerization, the phase inversion method can be used to create MIP-sensors [22]. The method consists in the use of ready-made polymers that are deposited on the surface of the working electrode from a solution in the presence of the analyte. Its main advantage is the simplicity and faster manufacturing of the MIP- sensor. To dissolve the two components, a solvent is chosen that is compatible with both the main polymer and the tem- plate. Their mixing makes it possible to form a "guest-host" complex in solution. MIP can be obtained in two ways: 1) wet phase inversion (WPI) – by adding another solvent, which causes the precipitation of the polymer associated with the template; 2) dry phase inversion (DPI) – the MIP- membrane is obtained by evaporating the solvent from the polymer during the heating process [23]. The DPI method is simpler and more convenient to manufacture, since, un- like WPI, it does not require the selection of a second sol- vent. WPI is also complicated by the fact that the template may not be deposited in the polymer composition or may be deposited in a very small amount, which leads to a low spec- ificity of the resulting MIP. DPI is usually carried out with heat. Therefore, in this case, polyarylenephthalides (PAP) [24–26], which are electrically conductive in thin layers and chemically resistant to heat and aggressive media, are of considerable interest and can be used. PAPs were previ- ously studied and applied in the manufacture of sensors for the creation of composite materials [15]. The selectivity and sensitivity of MIP-sensors are their main and most important characteristics. However, the deposition of a polymer on the electrode surface often leads to a decrease in currents due to an increase in resistance. Therefore, components that increase electrical conductivity must be added to the sensor layer. Recently, nanomaterials have been used for these purposes, such as Au and Pt nano- particles, single-walled and multi-walled carbon nano- tubes, reduced graphene oxide (rGO) [13, 17, 27], etc. At the same time, rGO is also used as a convenient matrix for im- mobilizing various components when creating efficient sen- sor platforms based on composite materials and increasing the sensitivity and selectivity of voltammetric sensors due to its unique properties, such as high electrical conductiv- ity, large specific surface area, mechanical strength, etc. Lincomycin hydrochloride (Lin) (Figure 1a), derived from Streptomyces lincolnensis, is a well-established antibiotic. It is active against most common Gram-positive bacteria, in- hibits cell growth and microbial protein synthesis, and is used to treat many infectious diseases [28] (staphylococcal, streptococcal, bacteroid infections, pneumonia, anthrax, fu- runculosis, carbuncles, impetigo, burns and wounds). In this work, to determine Lin, a voltammetric sensor based on a glassy carbon electrode (GCE) modified with rGO and PAP containing diphenylenethio- and diphenyleneoxide fragments in the main chain of the polymer in 1:1 ratios (Figure 1b) was developed. The characteristics of the sensor were studied using FTIR spectroscopy, electrochemical im- pedance spectroscopy (EIS), and cyclic voltammetry (CV). Lin was determined by differential pulse voltammetry (DPV). The analytical characteristics of the developed sen- sors, such as sensitivity, selectivity, linear range of concen- trations and limit of detection (LOD), were studied. 2. Experimental 2.1. Materials and reagents Lin (≥99.5%), K3Fe(CN)6 (≥99.0%) and K4Fe(CN)6 (≥99.0%), GO powder (15–20 sheets, 4–10% edge oxida- tion) were purchased from Sigma-Aldrich (USA). Samples of the PAP polymer (spectroscopically pure) were provided by the Laboratory for the Synthesis of Functional Polymers, Ural Federal Research Center, Russian Academy of Sciences (Ufa, Russia) [24–26]. The supporting electrolyte for Lin was a phosphate buffer solution (PBS, KH2PO4 + Na2HPO4, 0.1 M, pH 6.9). A 0.5 mM Lin solution was prepared by dis- solving an accurate weighed portion of the reagent in 25 mL of PBS. Solutions of lower concentrations were prepared by serial dilution. Urine samples were obtained with the writ- ten consent of the donor, blood plasma was purchased from the Ufa Republican Blood Transfusion Station. Urine and blood plasma samples, 2.5 ml in volume, were centrifuged for 10 min and diluted 10 times with PBS pH 6.9. Known amounts of Lin were added to the resulting solutions to ob- tain solutions with concentrations of 0.35 and 0.04 mM. All solutions were prepared using ultrapure deionized water with a specific electrical conductivity of 0.1 μS cm–1. Figure 1 Structure of Lin (a) and PAP polymer (b). https://doi.org/10.15826/chimtech.2023.10.2.10 https://doi.org/10.15826/chimtech.2023.10.2.10 Chimica Techno Acta 2023, vol. 10(2), No. 202310210 ARTICLE 3 of 8 DOI: 10.15826/chimtech.2023.10.2.10 All electrochemical measurements were performed on Autolab PGSTAT 204 potentiostat-galvanostat with an FRA32M impedance module (Metrohm Autolab Ins., Neth- erlands) with NOVA software. A standard three-electrode cell consisted of a modified GCE (Metrohm Autolab Ins., Netherlands) with a diameter of 3 mm as a working elec- trode, a platinum plate as an auxiliary electrode, and a sil- ver chloride electrode with a 3.5 M KCl solution as a refer- ence electrode. The pH of solutions was measured using a Seven Compact pH/Ion S220 pH meter (Mettler-Toledo AG, Switzerland). The solutions were stirred using an MR Hei- Tec magnetic stirrer (Heidolph, Germany). FTIR spectra of GO and rGO were recorded in the range 450–3500 cm–1 on an FTIR-8400S spectrometer (Shimadzu, Japan) at room temperature (resolution 8 cm–1, number of scans 30) with IR solution software. 2.2. Modification of the electrode surface To modify the GCE, 4 mg of GO was added to 1 ml of an aqueous ethanol solution (at a ratio of C2H5OH:H2O 1:1), af- ter which it was sonicated for 1 hour. GO dispersed in an aqueous ethanol solution was applied dropwise onto a care- fully polished GCE surface and dried under an IR lamp. The reduction of GO was carried out in a potentiostatic mode at a potential of E = –0.8 V for 3 minutes in a phosphate buffer solution. The conditions of GO reduction were chosen ex- perimentally according to the values of Lin oxidation cur- rents and RSD. To prepare mixtures of polymers with a tem- plate, 10 mg of the polymer and 5 mg of Lin were dissolved in 10 ml of N,N-Dimethylformamide (DMF). The poly- mer:template ratio was experimentally found to be optimal according to the Ip and relative standard deviation (RSD) values. The resulting polymer solutions with the template were applied to the GCE surface modified with rGO, fol- lowed by removal of the template in 1 M NaOH solution for 120 s with stirring. A sensor with a non-imprinted polymer (NIP) was obtained by a similar procedure, but without the addition of a template. 2.3. Experimental techniques Differential pulse voltammograms (DPV) were recorded in the potential range from 0 to 1.2 V with a scan rate of 20 mV s–1. Electrochemical impedance spectra were rec- orded in the frequency range from 500 kHz to 0.1 Hz with an amplitude of 10 mV. Cyclic voltammograms were rec- orded in the potential range from 0 to 1.3 V with a scan rate of 0.1 mV s–1. Before recording the DPV, the sensor was kept in the analyte solution for 80 s to incubate Lin. All meas- urements were carried out at a temperature of 25±0.1 °C. 3. Results and Discussion 3.1. FTIR and EIS results As a rule, rGO is used in electrochemical sensors to remove carboxyl and carbonyl groups and increase the electrical conductivity of the material. The FTIR spectrum (Figure 2) of GO shows characteristic bands at 1025, 1223, 1414, 1715, and 3419 cm–1, which can be attributed to the stretching vi- brations C–O, C–O–C, C–OH, C=O and –OH, while on rGO these characteristic bands sharply decrease or disappear, confirming that GO is reduced to rGO. The stretching vibra- tions at 1640 cm–1, observed both in GO and rGO, corre- spond to the C=C bonds present in the graphene sheet. The obtained FTIR spectra agree with the literature data [29]. The characteristic bands in the Lin spectrum are those at 1657 and 1567 cm–1, corresponding to the C=O stretching vibrations and N–H bending vibrations of the amide group, respectively. Stretching of S–CH3 is observed at 1107 cm–1, N–H – at 1041 cm–1, C–O–C (ether bond) – at 1263 cm–1, C–H (aliphatic) – at 2955 cm–1. A wide band of O–H stretch- ing vibrations with a complex contour is observed at 3528– 3289 cm–1, which, moreover, overlaps the N–H stretching vibrations. The characteristic bands in the PAP spectrum are 1770 and 1078 cm–1, corresponding to the C=O and C–O–C bonds of the phthalide group, 757 cm–1 corresponds to the Ar–S–Ar bond, 1244 cm–1 – to the Ar–O–Ar bonds. The characteristic bands of Lin appear in the spectrum of the PAP-Lin complex. And the band at 1770 cm–1, corresponding to C=O, shifts to 1758 cm–1, which may be due to the partic- ipation of this group in the formation of a hydrogen bond. EIS with [Fe(CN)6]3–/4– showed that each modification stage has a different effect on the currents of the [Fe(CN)6]3–/4– redox pair. The lowest resistance to electron transfer Ret was observed on GCE/rGO (Figure 3a, curve 3), while on unreduced GO (curve 2) the resistance is higher. When a PAP polymer film is deposited on GCE/rGO, the re- sistance increases (curve 4), but it does not significantly exceed the resistance on bare GCE, which is due to the elec- trical conductivity of the PAP. After Lin is washed out of the polymer, Ret decreases (curve 5) and the electron transfer rate increases due to the formation of pores in polymers through which [Fe(CN)6]3–/4– ions penetrate. Figure 2 FTIR spectra of GO (1), rGO (2), Lin (3), PAP (4), PAP-Lin (5). https://doi.org/10.15826/chimtech.2023.10.2.10 https://doi.org/10.15826/chimtech.2023.10.2.10 Chimica Techno Acta 2023, vol. 10(2), No. 202310210 ARTICLE 4 of 8 DOI: 10.15826/chimtech.2023.10.2.10 Figure 3 Nyquist diagrams of GCE (1), GCE/GO (2), GCE/rGO (3), GCE/rGO/PAP (4), GCE/rGO/miPAP (5) in 5 mM [Fe (CN)6] 3–/4– (1:1, 0.1 M KCl, 0.1 V s–1) (a); CV of the supporting electrolyte on GCE/rGO/miPAP before (1) and after (2) washing out of Lin from the polymer film in the supporting electrolyte (pH 6.9, 0.1 V s–1) (b). Also, Figure 3b shows that the CVs obtained in the sup- porting electrolyte solution before and after Lin was washed out from the polymer film differ: curve 1 shows a small peak of Lin oxidation due to the presence of Lin in the polymer film, which disappears after washing out in a 1 M NaOH solution with stirring for 120 s (Figure 3b, curve 2), confirming the effectiveness of the chosen technique. 3.2. Quantum-chemical modeling of the interac- tion of Lin with the PAP polymer Based on the structure of Lin, hydroxyl groups in the galac- topyranose fragment can be identified as possible centers of interaction, which suggests a tendency to form hydrogen bonds. The lactone fragments of the phthalide blocks of the polymer contain oxygen atoms, which can act as hydrogen bond acceptors. To calculate possible interactions, the fol- lowing simplification was introduced: two blocks consist- ing of two units were used as a polymer. After the initial optimization of the structures by the AM1 semi-empirical method, possible complexes were calculated by the CHARMM method [30] in the HyperChem program (Figure 4). The possibility of hydrogen bonding (2.017 Å, 2.668 Å and 2.713 Å) and the formation of cavities in the polymer for the antibiotic molecule was confirmed. 3.3. Lin detection Lin electrooxidation is a one-electron one-proton process with the formation of a dimerized product [6]. Figure 5 shows the DPV of a Lin solution on bare GCE, GCE/rGO, on GCE modified with PAP, and on GCE modified with molecu- larly imprinted PAP (miPAP). The deposition of GO on the surface of the GCE with subsequent electrochemical reduc- tion leads to an increase in the sensitivity of the sensor to Lin, as well as to a shift in the oxidation potential of Lin to the cathode region, which, apparently, is due to the facili- tation of the process of its oxidation on GO. The obtained DPVs of the Lin solution on GCE/rGO/miPAP are consistent with the previously obtained EIS data. The linear range of the dependence of Lin oxidation cur- rent on its content in the solution on GCE/rGO/miPAP re- mains in the concentration range from 2.5·10–7 to 5·10–4 M, with a detection limit of 6.8·10–8 M (Figure 6). Figure 4 Possible interactions between PAP-polymer (blue) and Lin (yellow) optimized by the quantum chemical modeling. Figure 5 DPVs of 0.5 mM Lin solution on GCE (1), GCE/rGO (2), GCE/rGO/miPAP (3), GCE/rGO/PAP (4) (PBS, 20 mV s–1). https://doi.org/10.15826/chimtech.2023.10.2.10 https://doi.org/10.15826/chimtech.2023.10.2.10 Chimica Techno Acta 2023, vol. 10(2), No. 202310210 ARTICLE 5 of 8 DOI: 10.15826/chimtech.2023.10.2.10 In addition, Figure 6b shows the calibration curves ob- tained at each stage of the GCE modification. The corre- sponding equations are as follows: y = (2.99±0.16)x + (0.27±0.02), R² = 0.9966, (1) y = (6.21x±0.26) + (0.88±0.04), R² = 0.9914, (2) y = (1.63±0.13)x + (0.12±0.03), R² = 0.9971, (3) y = (4.97±0.21)x + (0.37±0.02), R² = 0.9975. (4) It can be seen that in all curves there is a linear depend- ence of the peak current on the Lin concentration. The se- lectivity coefficients S on the GCE are lower than those on the final sensor. It should be noted that the sensitivity on GCE/rGO is higher than that on GCE/rGO/miPAP, which is associated with an increase in resistance when using the PAP-polymer, being consistent with the EIS data. However, the resulting S and LOD on GCE/rGO/miPAP is sufficient to detect Lin in real samples [31–33]. The linear dependences on GCE/rGO/PAP and GCE/rGO/miPAP show that the pres- ence of molecular imprints increases the sensitivity of GCE/rGO/miPAP by a factor of 3.05 (SMIP/SNIP). These re- sults illustrate the high sensitivity and selectivity of the GCE/rGO/miPAP sensor. The obtained results show that the developed sensor for Lin detecting is comparable to the electrochemical sen- sors described in the literature, are not inferior to them in their characteristics, and sometimes even surpass them (Table 1). This confirm the good sensitivity of GCE/rGO/miPAP in Lin detection. It should be noted that the sensor fabrication procedure has a simple strategy and lower number of steps for creating MIP due to the use of the phase inversion method; as a result, this approach is more express. Other methods listed in Table 1 such as elec- trochemiluminescence, surface plasmon resonance, Ra- man spectroscopy, colorimetric, photoelectrochemical methods can detect Lin with a lower LOD. However, ac- cording to [31–33], the concentration of Lin and its ana- logues in biological fluids is 0.25–16 μg mL–1 (i.e. 1.2·10–6– 3.9·10–5 M). Thus, the LOD of the developed sensor is suf- ficient to detect Lin. To estimate the correctness of the detection of Lin, the “spike-recovery” test was used (Table 2). The sensor made it possible to detect the Lin concentration with high accu- racy over the entire linear range; the RSD did not exceed 3.6%, which indicates good reproducibility of the detection, and the values of the relative measurement error not ex- ceeding 3% indicate the accuracy of the results. To assess the analytical capabilities of the proposed sensor, it was used to detect Lin in human urine and blood plasma. The RSD did not exceed 7.5%, and the recovery was 93–108%. Statistical evaluation of the measurement results by the "spike-recovery" test indicates the absence of a significant systematic error. The repeatability and stability tests of the GCE/rGO/miPAP were carried out for 5·10–4 mM Lin. After 10 successive assays, the response signal of the GCE/rGO/miPAP still remained up to 97.1% of its initial val- ues with RSD 3.9%. After 10 days of storage at room tem- perature, the current responses of the GCE/rGO/miPAP re- mained up to 95.3% of its initial value with RSD 4.1%. 4. Limitations When recording the DPV, the capacitive currents on the GCE and the GCE modified with rGO differ significantly. In this regard, it is necessary to carry out the baseline correction, as well as the curve smoothing based on the Savitzky-Golay algorithm. Figure 6 DPVs of Lin solutions of various concentrations (1 – 0.0025, 2 – 0.01, 3 – 0.03, 4 – 0.05, 5 – 0.1, 6 – 0.2, 7 – 0.3, 8 – 0.4, 9 – 0.5 mM) on GCE/rGO/miPAP (a); calibration curves of GCE (1), GCE/rGO (2), GCE/rGO/PAP (3) and GCE/rGO/miPAP (4) sensors (PBS, 20 mV s–1, n = 5, P = 0.95) (b). https://doi.org/10.15826/chimtech.2023.10.2.10 https://doi.org/10.15826/chimtech.2023.10.2.10 Chimica Techno Acta 2023, vol. 10(2), No. 202310210 ARTICLE 6 of 8 DOI: 10.15826/chimtech.2023.10.2.10 Table 1 Figures of merit of proposed sensor GCE/rGO/miPAP in comparison with the reported electrochemical sensors and other meth- ods used for the Lin detection. Sensing element Technique Linear range, M LOD, M References Electrochemical sensors GCE/rGO/miPAP DPV 2.5·10–7–5·10–4 6.8·10–8 This work MWNTs-DHP/GCE CV 4.5·10–7–1.5·10–4 2.0·10–7 [5] Au DPV 8.0·10–6–1.0·10–4 1.7·10–7 [6] BDD thin film electrodes CV 5.0·10–7–1.3·10–4 2.0·10–8 [34] DME in KH2PO4-Na2HPO4-K2S2O8 solution CV 8.5·10 –8–9.0·10–5 4.0·10–8 [35] Au-PtNPs/nanoPAN/CS/GCE CV 7.4·10–6–2.5·10–4 2.5·10–6 [36] Other methods MIP-Au-GO/GCE ECL 5.0·10–12–1.0·10–9 1.6·10–13 [37] SnO2/chitosan/g-C3N4/GCE ECL 2.5·10 –10–2.5·10–7 6.9·10–11 [38] N-doped Ti3C2 QDs/BiOBr PEC 1.0·10 –14–1.0·10–8 3.6·10–15 [39] PFCs-TiO2/NG/ITO- ZnPc/MoS2/ITO PEC 1.0·10 –11–1.0·10–5 3.3·10–12 [40] HAuCl4/NaOH solution Colorimetry 1.0·10 –6–2.5·10–5 9.7·10–7 [41] Au-Pt NR-apt/cDNA/PTP/Eu MOF/GCE ECL-SPR 2.5·10–10–2.5·10–4 6.4·10–11 [42] mAb-AuNPs-DTNB SERS 2.5·10–13–2.5·10–9 7.1·10–13 [43] CdS QDs/C-g-C3N4 ECL 1.2·10 –11–2.5·10–4 4.9·10–11 [44] MWNTs – multi-wall carbon nanotubes; DHP – dihexadecylphosphate; BDD – boron-doped diamond; DME – dropping mercury electrode; Au-PtNPs – Au-Pt alloy nanoparticles; nanoPAN – polyaniline nanotube; CS – chitosan; g-C3N4 – graphene-like carbon nitride; QDs – quantum dots; TiO2/NG – nitrogen-doped graphene-loaded TiO2 nanoparticles; PFC – dualphotoelectrode photofuel cell; ZnPc/MoS2 – zinc phthalocyanine nanoparticles sensitized MoS2; ITO – indium tin oxide; NR – nanorod; apt/cDNA – bridge of aptamer and comple- mentary DNA; PTP – PTCA (perylene tetracarboxylic dianhydride) – PANI (polyanilin); Eu MOF – europium metal–organic framework; AuNPs – gold nanoparticles; DTNB – 5,5′-dithiobis (2-nitrobenzoic acid); mAb – monoclonal antibody; C-g-C3N4 – carboxylated g-C3N4; ECL – elec- trochemiluminescence; PEC – photoelectrochemical; SPR – surface plasmon resonance; SERS – surface-enhanced Raman spectroscopy. Table 2 Lin determination using DPV on the GCE/rGO/miPAP (PBS, 20 mV s–1, n = 5, P = 0.95). Spiked, µM Found, µM RSD, % Recovery, % Lin solution 350 353±9 1.9 101 40 41±6 2.7 103 Lin in urine 350 346±15 6.6 99 40 43±5 5.2 108 Lin in blood plasma 350 343±11 7.5 98 40 37±6 5.3 93 5. Conclusions Thus, to detect the antibiotic Lin, we developed the sensor based on GCE modified with rGO and molecularly im- printed PAP obtained by phase inversion by solvent evap- oration. It should be noted that when the phase inversion method is used to obtain the MIP, the sensor manufactur- ing process is greatly simplified. It was shown that the de- veloped sensor has a high selectivity for the detected an- tibiotic, and the presence of specific binding sites in the polymer film makes it possible to detect Lin with a sensi- tivity that is 3.05 times higher than that of a similar sen- sor without molecular imprints. The resulting sensor was successfully used to determine Lin in biological fluids. ● Supplementary materials No supplementary materials are available. ● Funding This work was supported by the Russian Science Founda- tion (grant no. 21-73-00295, https://rscf.ru/en/project/21- 73-00295/). ● Acknowledgments The authors are grateful to the Laboratory for the Synthesis of Functional Polymers, Ural Federal Research Center, Rus- sian Academy of Sciences, supervised by Kraikin V.A. for providing polymer samples. ● Author contributions Conceptualization: V.N.M., Y.A.Y. Data curation: Y.A.Y. Formal Analysis: M.I.N., Y.A.Y., L.R.Z. Funding acquisition: Y.A.Y., D.A.D. Investigation: D.A.D., M.I.N. Methodology: V.N.M., Y.A.Y. Project administration: Y.A.Y. Resources: Y.A.Y. Supervision: V.N.M. Validation: D.A.D., Y.A.Y. Visualization: D.A.D., M.I.N., Y.A.Y., L.R.Z. Writing – original draft: Y.A.Y., M.I.N. Writing – review & editing: V.N.M. https://doi.org/10.15826/chimtech.2023.10.2.10 https://doi.org/10.15826/chimtech.2023.10.2.10 https://rscf.ru/en/project/21-73-00295/ https://rscf.ru/en/project/21-73-00295/ Chimica Techno Acta 2023, vol. 10(2), No. 202310210 ARTICLE 7 of 8 DOI: 10.15826/chimtech.2023.10.2.10 ● Conflict of interest The authors declare no conflict of interest. ● Additional information Author IDs: Yulia A. Yarkaeva, Scopus ID 56872864300; Daria A. Dymova, Scopus ID 57899452900; Marat I. Nazyrov, Scopus ID 57330245700; Liana R. Zagitova, Scopus ID 57201803011; Valery N. Maistrenko, Scopus ID 6603789725. Website: Ufa University of Science and Technology, https://uust.ru/page-contacts. References 1. Pham THY, Mai TT, Nguyen HA, Chu TTH, Vu TTH, Le QH. Voltammetric determination of amoxicillin using a reduced graphite oxide nanosheet electrode. J Anal Methods Chem. 2021;2021:8823452. doi:10.1155/2021/8823452 2. Valenga MGP, Felsner ML, de Matos CF, de Castro EG, Galli A. Development and validation of voltammetric method for de- termination of amoxicillin in river water. Anal Chim Acta. 2020;1138:79–88. doi:10.1016/j.aca.2020.09.020 3. Li H, Xu B, Wang D, Zhou Y, Zhang H, Xia W, Xu S, Li Y. Im- munosensor for trace penicillin G detection in milk based on supported bilayer lipid membrane modified with gold nano- particles. J Biotechnol. 2015;203:97–103. doi:10.1016/j.jbiotec.2015.03.013 4. Bougrini M, Florea A, Cristea C, Sandulescu R, Vocanson F, Errachid A, Bouchikhi B, Bari NE. Development of a novel sensitive molecularly imprinted polymer sensor based on electropolymerization of a microporous-metal-organic frame- work for tetracycline detection in honey. Food Control. 2016;59:424–429. doi:10.1016/j.foodcont.2015.06.002 5. Wu Y, Ye S, Hu S. Electrochemical study of lincomycin on a multi-wall carbon nanotubes modified glassy carbon elec- trode and its determination in tablets. J Pharm Biomed Anal. 2006;41:820–824. doi:10.1016/j.jpba.2006.01.037 6. Abbar JC, Meti MD, Nandibewoor ST. Anodic Voltammetric behavior of lincomycin and its electroanalytical determina- tion in pharmaceutical dosage form and urine at gold elec- trode. Z Phys Chem. 2017;231(5):957–970. doi:10.1515/zpch-2015-0745 7. Prado TM, Foguel MV, Gonçalves LM, Sotomayor MPT. β-Lac- tamase-based biosensor for the electrochemical determina- tion of benzylpenicillin in milk. Sens Actuators B Chem. 2015;210:254–258. doi:10.1016/j.snb.2014.12.108 8. Samanidou V, Nisyriou S Multi-residue methods for confirm- atory determination of antibiotics in milk. J Sep Sci. 2008;31(11):2068–2090. doi:10.1002/jssc.200700647 9. Mohsenzadeh MS, Mohammadinejad A, Mohajeri SA. Simple and selective analysis of different antibiotics in milk using molecularly imprinted polymers: a review. Food AdditCon- tam Part A. 2018;35(10):1959–1974. doi:10.1080/19440049.2018.1508889 10. Bitas D, Samanidou V. Molecularly imprinted polymers as ex- tracting media for the chromatographic determination of an- tibiotics in milk. Molec. 2018;23(2):316. doi:10.3390/molecules23020316 11. Kennedy DG, McCracken RJ, Cannavan A, Hewitt SA. Use of liquid chromatography–mass spectrometry in the analysis of residues of antibiotics in meat and milk. J Chromatogr A. 1998;812(1–2):77–98. doi:10.1016/S0021-9673(98)00048-X 12. Cañada-Cañada F, Muñoz de la Peña A, Espinosa-Mansilla A. Analysis of antibiotics in fish samples. Anal Bioanal Chem. 2009;395(4):987–1008. doi:10.1007/s00216-009-2872-z 13. Yarkaeva Y, Maistrenko V, Dymova D, Zagitova L, Nazyrov M. Polyaniline and poly(2-methoxyaniline) based molecular im- printed polymer sensors for amoxicillin voltammetric deter- mination. Electrochim Acta. 2022;433:141222. doi:10.1016/j.electacta.2022.141222 14. Zagitova L, Maistrenko V, Yarkaeva Y, Zagitov V, Zilberg R, Kovyazin P, Parfenova L. Novel chiral voltammetric sensor for tryptophan enantiomers based on 3-neomenthylindene as recognition element. J Electroanal Chem. 2021;880:114939. doi:10.1016/j.jelechem.2020.114939 15. Yarkaeva Y, Maistrenko V, Zagitova L, Nazyrov M, Berestova T. Voltammetric sensor system based on Cu(II) and Zn(II) amino acid complexes for recognition and determination of atenolol enantiomers. J Electroanal Chem. 2021;903:115839. doi:10.1016/j.jelechem.2021.115839 16. Yarkaeva Y, Islamuratova E, Zagitova L, Gus’kov V, Zil’berg R, Maistrenko V. A Sensor for the recognition and determination of tryptophan enantiomers based on carbon-paste electrode modified by enantiomorphic crystals of bromotriphenylme- thane. J Anal Chem. 2021;76(11):1345–1354. doi:10.1134/S1061934821110162 17. Zagitova L, Yarkaeva Y, Zagitov V, Nazyrov M, Gainanova S, Maistrenko V. Voltammetric chiral recognition of naproxen enantiomers by N-tosylproline functionalized chitosan and reduced graphene oxide based sensor. J Electroanal Chem. 2022;992:116774. doi:10.1016/j.jelechem.2022.116744 18. Benachio I, Lobato A, Goncalves LM. Employing molecularly imprinted polymers in the development of electroanalytical methodologies for antibiotic determination. J Mol Recognit. 2021;34:2878. doi:10.1002/jmr.2878 19. Wulff G. Fourty years of molecular imprinting in synthetic polymers: origin, features and perspectives. Microchim Acta. 2013;180(15–16):1359–1370. doi:10.1007/s00604-013-0992-9 20. Baeza-Fonte AN, Garcés-Lobo I, Luaces-Alberto MD, Gon- çalves LM, Sotomayor M, Valdés-González AC. Determination of cephalosporins by UHPLC-DAD using molecularly im- printed polymers. J Chromatogr Sci. 2018;56(2):187–197. doi:10.1093/chromsci/bmx099 21. Alenazi NA, Manthorpe JM, Lai EPC. Selectivity enhancement in molecularly imprinted polymers for binding of bisphenol A. Sensors. 2016;16(10):1697. doi:10.3390/s16101697 22. Gavrila AM, Stoica EB, Iordache TV, Sârbu A. Modern and dedicated methods for producing molecularly imprinted poly- mer layers in sensing applications. Appl Sci. 2022;12:3080. doi:10.3390/app12063080 23. BelBruno J. Molecularly imprinted polymers. Chem Rev. 2019;119:94–119. doi:10.1021/acs.chemrev.8b00171 24. Kraikin V, Fatykhov A, Gileva N, Kravchenko A, Salazkin S. NMR study of dyadic and triadic splitting in copoly(ar- ylene)phthalides based on diphenyl oxide and diphenyl sul- fide. Magn Reson Chem. 2020;59(1):61–73. doi:10.1002/mrc.5079 25. Salazkin S, Shaposhnikova V, Machulenko L, Gileva N, Krai- kin V, Lachinov A. Synthesis of polyarylenephthalides pro- spective as smart polymers. Polym Sci Ser A. 2008;50(3):243–259. doi:10.1134/S0965545X08030024 26. Gileva N, Kraikin V, Sedova E, Lobov M, Kuznetsov S, Sala- zkin S. Control over the composition and microstructure of copoly(arylene phthalides). Russ J Appl Chem. 2005;78(10):1683–1686. 27. Salikhov R, Zilberg R, Mullagaliev I, Salikhov T, Teres Y. Nanocomposite thin film structures based on polyaryleneph- thalide with SWCNT and graphene oxide fillers. Mendeleev Commun. 2022;32:520–522. doi:10.1016/j.mencom.2022.07.029 28. Hardman JG, Limbird LE, Gilman AG. Goodman & Gilman’s the pharmacological basis of therapeutics. 10th Edition. New York: McGraw-Hill; 2001. 256 p. https://doi.org/10.15826/chimtech.2023.10.2.10 https://doi.org/10.15826/chimtech.2023.10.2.10 http://www.scopus.com/inward/authorDetails.url?authorID=56872864300&partnerID=MN8TOARS http://www.scopus.com/inward/authorDetails.url?authorID=57899452900 http://www.scopus.com/inward/authorDetails.url?authorID=57330245700 http://www.scopus.com/inward/authorDetails.url?authorID=57201803011 http://www.scopus.com/inward/authorDetails.url?authorID=6603789725 https://uust.ru/page-contacts https://doi.org/10.1155/2021/8823452 https://doi.org/10.1016/j.aca.2020.09.020 https://doi.org/10.1016/j.jbiotec.2015.03.013 https://doi.org/10.1016/j.foodcont.2015.06.002 https://doi.org/10.1016/j.jpba.2006.01.037 https://doi.org/10.1515/zpch-2015-0745 https://doi.org/10.1016/j.snb.2014.12.108 https://doi.org/10.1002/jssc.200700647 https://doi.org/10.1080/19440049.2018.1508889 https://doi.org/10.3390/molecules23020316 https://doi.org/10.1016/S0021-9673(98)00048-X https://doi.org/10.1007/s00216-009-2872-z https://doi.org/10.1016/j.electacta.2022.141222 https://doi.org/10.1016/j.jelechem.2020.114939 https://doi.org/10.1016/j.jelechem.2021.115839 https://doi.org/10.1134/S1061934821110162 https://doi.org/10.1016/j.jelechem.2022.116744 https://doi.org/10.1002/jmr.2878 https://doi.org/10.1007/s00604-013-0992-9 https://doi.org/10.1093/chromsci/bmx099 https://doi.org/10.3390/s16101697 https://doi.org/10.3390/app12063080 https://doi.org/10.1021/acs.chemrev.8b00171 https://doi.org/10.1002/mrc.5079 https://doi.org/10.1134/S0965545X08030024 https://doi.org/10.1016/j.mencom.2022.07.029 Chimica Techno Acta 2023, vol. 10(2), No. 202310210 ARTICLE 8 of 8 DOI: 10.15826/chimtech.2023.10.2.10 29. Luo H, Li H, Ge Q, Cong H, Tao Z, Liu M. An electrochemical sensor for enantio-recognition of tyrosine based on a chiral macrocycle functionalized rGO. Microchem J. 2021;164:105949. doi:10.1016/j.microc.2021.105949 30. Wu YY, Zhao FQ, Ju XH, A comparison of the accuracy of semi-empirical PM3, PDDG and PM6 methods in predicting heats of formation for organic compounds. J Mex Chem Soc. 2014;58:223–229. doi:10.29356/jmcs.v58i2.182 31. Girondi NG, Barreto F, Pigatto MC, Dalla Costa T. Sensitive analytical method to quantify clindamycin in plasma and mi- crodialysate samples: Application in a preclinical pharmaco- kinetic study. J Pharm Biomed Anal. 2018;153:57–62. doi:10.1016/j.jpba.2018.02.005 32. Hu Y, Zhu Q, Wang Y, Liao C, Jiang G. A short review of hu- man exposure to antibiotics based on urinary biomonitoring. Sci Total Environ. 2022;830:154775. doi:10.1016/j.scitotenv.2022.154775 33. Gouri SS, Venkatachalam D, Dumka VK. Pharmacokinetics of lincomycin following single intravenous administration in buffalo calves. Trop Anim Health Prod. 2014;46:1099–1102. doi:10.1007/s11250-014-0595-4 34. Boonsong K, Chuanuwatanakul S, Wangfuengkanagul N, Chailapakul O. Electroanalysis of lincomycin using boron- doped diamond thin film electrode applied to flow injection system. Sensors Actuators B. 2005;108:627–632. doi:10.1016/j.snb.2004.12.087 35. Li N, Song J, Wei Guo, Xu M. Study and application of parallel catalytic hydrogen wave of lincomycin in the presence of per- sulfate. Microchem J. 2004;77:23–28. doi:10.1016/j.microc.2003.10.001 36. Wang X, Yang T, Jiao K. Electrochemical study of lincomycin on Au-PtNPs/nanoPAN/ Chitosan nanocomposite membrane and its determination in injections. Chem Res Chin Univ. 2010;26(3):371–375. 37. Li S, Liu C, Yin G, Zhang Q, Luo J, Wu N. Aptamer-molecularly imprinted sensor base on electrogenerated chemilumines- cence energy transfer for detection of lincomycin. Biosensors Bioelectron. 2017;91:687–691. doi:10.1016/j.bios.2017.01.038 38. Liu X-P, Huang B, Mao C-J, Chen J-S, Jin B-K. Electrochemilu- minescence aptasensor for lincomycin antigen detection by using a SnO2/chitosan/g-C3N4 nanocomposite. Talanta. 2021;233:122546. doi:10.1016/j.talanta.2021.122546 39. Wei M, Du X, Zhang Y, Shan X, Wang W, Chen Y, Jiang D, Xu F, Shiigi H, Chen Z. Ultrasensitive self-driven photoelectro- chemical aptasensor for lincomycin detection based on oxy- gen vacancy-tunable BiOBr nanosheet coupled with dual- function of N-doped Ti3C2 quantum dots. Biosensors Bioelec- tron. 2022;12:100266. doi:10.1016/j.biosx.2022.100266 40. Wen Z, Ding L, Zhang M, You F, Yuan R, Wei J, Qian J, Wang K. A membrane/mediator-free high-power density dual-pho- toelectrode PFC aptasensor for lincomycin detection in milk and chicken. Anal Chim Acta. 2023;1245:340880. doi:10.1016/j.aca.2023.340880 41. Leng Y, Hu F, Ma C, Du C, Ma L, Xu J, Lin Q, Sang Z, Lu Z. Simple, rapid, sensitive, selective and label-free lincomycin detection by using HAuCl4 and NaOH. RSC Adv. 2019;9:28248–28252. doi:10.1039/C9RA04095A 42. Li J, Luo M, Jin C, Zhang P, Yang H, Cai R, Tan W. Plasmon- Enhanced Electrochemiluminescence of PTP-Decorated Eu MOF-Based Pt-tipped au bimetallic nanorods for the lincomy- cin Assay. Eur Food Res Technol. 2022;248:2157–2165. doi:10.1021/acsami.1c21528 43. Shi Q, Tao C, Kong D. Multiplex SERS‑based lateral flow assay for one‑step simultaneous detection of neomycin and linco- mycin in milk. Eur Food Res Technol. 2022;248:2157–2165. doi:10.1007/s00217-022-04038-3 44. Fan Y, Liu Z, Wang J, Cui C, Hu L. An “off–on” electrochemilu- minescence aptasensor for determination of lincomycin based on CdS QDs/carboxylated g-C3N4. Microchim Acta. 2023;190(1):11. doi:10.1007/s00604-022-05587-w https://doi.org/10.15826/chimtech.2023.10.2.10 https://doi.org/10.15826/chimtech.2023.10.2.10 https://doi.org/10.1016/j.microc.2021.105949 https://doi.org/10.29356/jmcs.v58i2.182 https://doi.org/10.1016/j.jpba.2018.02.005 https://doi.org/10.1016/j.scitotenv.2022.154775 https://doi.org/10.1007/s11250-014-0595-4 https://doi.org/10.1016/j.snb.2004.12.087 https://doi.org/10.1016/j.microc.2003.10.001 https://doi.org/10.1016/j.bios.2017.01.038 https://doi.org/10.1016/j.talanta.2021.122546 https://doi.org/10.1016/j.biosx.2022.100266 https://doi.org/10.1016/j.aca.2023.340880 https://doi.org/10.1039/C9RA04095A https://doi.org/10.1021/acsami.1c21528 https://doi.org/10.1007/s00217-022-04038-3 https://doi.org/10.1007/s00604-022-05587-w