Electrochemical creatinine determination with metal-organic framework based on copper and acetylenedicarboxylic acid as catalyst published by Ural Federal University eISSN 2411-1414 chimicatechnoacta.ru LETTER 2023, vol. 10(2), No. 202310201 DOI: 10.15826/chimtech.2023.10.2.01 1 of 6 Electrochemical creatinine determination with metal- organic framework catalyst based on copper and acetylenedicarboxylic acid Andrei V. Okhokhonin * , Alsu A. Ibatullina , Yulia V. Izmozherova , Marina I. Stepanova , Anatoly I. Matern , Alisa N. Kozitsina Institute of Chemical Engineering, Ural Federal University, Ekaterinburg 620009, Russia * Corresponding author: a.v.ohohonin@urfu.ru This paper belongs to a Regular Issue. Abstract Fast and accurate determination of creatinine is critical in kidney function diagnostics. This paper discusses the usage of the metal-organic frame- work based on copper(II) and acetylenedicarboxylic acid (CuADCA) as a catalyst of electrochemical oxidation of creatinine, glucose and urea. CuADCA was synthesized by deprotonation with triethylamine for the first time. Successful synthesis was confirmed by FTIR and EDS. The material was characterized by SEM, EIS, and CV. CuADCA forms laminated-like flakes with diameter from 1 µm to 20 µm, which is consistent with the polymer-like structure. CV and EIS analyses showed that CuADCA immo- bilized on GCE acts as a catalyst in electrooxidation reaction of glucose, urea, and creatinine. The sensitivity of creatinine detection, 1057±99 µA/mM, was higher than the detection sensitivity of glucose and urea by more than 100 times with the limit of detection of 2 µM, so CuADCA is a promising material for further development of enzymeless sensors for creatinine. Keywords metal-organic framework creatinine voltammetry electrochemical catalyst enzymeless determination Received: 03.03.23 Revised: 18.03.23 Accepted: 19.03.23 Available online: 29.03.23 © 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 The determination of creatinine in urine and blood is criti- cal in case of diagnosis of kidney and liver diseases. Modern methods for these purposes are chromatography, mass- spectrometry, and Jaffe reaction. Chromatography and mass-spectrometry require cumbersome devices highly qualified personnel and high-cost consumables. The Jaffe reaction, despite its simplicity and availability, is associ- ated with using poisonous and explosive picric acid and low specificity, causing erroneously high creatinine results in the presence of proteins, glucose, acetoacetate, ascorbic acid, guanidine, acetone, cephalosporins, aminoglycosides, ketone bodies, α-keto acids, and other organic compounds. Thereby, the development of new simple, express, safe, and specific methods of creatinine determination is an urgent task in the field of medical diagnosis. A promising approach for solving this problem involves electrochemical methods, especially voltammetry, due to its accuracy, high rate and low-cost of consumables and de- vices. However, analysis of organic compounds, including creatinine, on traditional electrodes (platinum, glassy carbon, etc.) is difficult because of the absence of inherent electroactivity. There are a few ways of avoiding this draw- back. One of them is using an electrochemical catalysts, which can reversibly oxidize or reduce and transfer elec- trons from non-electroactive molecule to electrode (or vice versa). The most popular electrochemical catalysts used in electrochemical sensors are metal (platinum, gold, palla- dium, etc.) and carbon nanomaterials, metal–organic com- plexes. During the past two decades, metal–organic frame- works (MOFs), also known as porous coordination poly- mers (PCPs), have experienced explosive growth. MOFs ex- hibit a wide variety of potential applications in catalysis, gas storage and separation, luminescence, and drug deliv- ery, owing to their specific features, such as structural di- versity, flexibility and tailorability, high porosity, large sur- face area, and extraordinary adsorption affinities [1]. In electrochemical biosensing MOFs were used for determina- tion of cancer biomarkers [2], heavy metals [3], herbicides [4], H2O2 [5], bisphenol A [6] and other analytes. There are several MOFs that are used to absorb creati- nine in hemodialysis apparatus [7] and to detect creatinine: in the works [8, 9] the authors report a luminescent sensor http://chimicatechnoacta.ru/ https://doi.org/10.15826/chimtech.2023.10.2.01 mailto:a.v.ohohonin@urfu.ru http://creativecommons.org/licenses/by/4.0/ https://orcid.org/0000-0003-2922-7623 https://orcid.org/0009-0003-2562-9606 https://orcid.org/0009-0008-3601-5016 https://orcid.org/0000-0002-1779-1756 https://orcid.org/0009-0000-4645-0961 https://orcid.org/0000-0002-0263-2111 https://crossmark.crossref.org/dialog/?doi=https://doi.org/10.15826/chimtech.2023.10.2.01&domain=pdf&date_stamp=2023-03-29 Chimica Techno Acta 2023, vol. 10(2), No. 202310201 LETTER 2 of 6 DOI: 10.15826/chimtech.2023.10.2.01 for detecting creatinine based on lanthanide–organic frameworks, but such sensors are non-reusable and require expensive equipment. Anyway, electrochemical creatinine sensors based on MOFs have not been described in the lit- erature. In the past, a lot of MOFs with different linkers and metal ions were synthesized and applied as sensing recep- tors in electrochemical sensors. Significant portion of these MOFs are based on copper(I) or (II) ions because of their high catalytic activity and low cost [1, 5, 6, 10–12]; besides, copper ions form a strong coordination bond with creati- nine: from 2 to 4 creatinine molecules per one Cu2+ ion [13]. A variety of MOF forms and their characteristics are available due to using different linkers between metal ions. The main requirements for these molecules are the rigid structure, e.g., imidazole, benzene ring(s), acetylene group, etc., and the presents of two or more functional groups that can attach ions (–COOH, –NH2, pyridines, etc.) [1]. One of the promising linkers for MOF synthesis is acetylenedicar- boxylic acid (ADCA), which meets all these requirements [14]. Some amount of copper and ADCA containing CPs were synthesized to date[14, 15]. So, the aims of the present work were the synthesis of Cu2+-acetylenedicarboxylic acid MOF (CuADCA) and the in- vestigation of its electrochemical behavior towards creati- nine in the presence of glucose and urea. 2. Materials and Methods 2.1. Materials CuCl2∙2H2O, ethyl alcohol, dimethylformamide (DMF), acet- ylenedicarboxylic acid (ADCA, 95%, Sigma-Aldrich, USA), triethylamine (TEA, Sigma, USA), creatinine (Millipore, Germany), glucose (PanReac, Spain), urea (Sigma-Aldrich, USA), Na2HPO4 (Sigma-Aldrich, USA), KH2PO4 (Sigma-Al- drich, USA), KCl (Sigma-Aldrich, USA), NaCl (Sigma-Al- drich, USA), K4[Fe(CN)6] (Sigma-Aldrich, USA), K3[Fe(CN)6] (Sigma-Aldrich, USA). Deionized water (18.2 MΩ) was produced with Barnstead™ Pacific TII Water Purification System (Germany) and was used in all experi- ments. Phosphate buffer saline (PBS) pH = 7.4 was pre- pared by dissolving of 8.00 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4 and 0.24 g of KH2PO4 in 1 liter of water with fur- ther adjustment to the desired pH. 2.2. Apparatus Voltammetric and electrochemical impedance spectroscopy (EIS) studies were performed using an Autolab PGSTAT302N potentiostat/galvanostat (Metrohm AG, The Netherlands). Three-electrode electrochemical cell was used: glassy carbon disk working electrode (GCE) with S=0.07 cm2, Ag/AgCl reference electrode and glassy carbon rod counter electrode (all – Metrohm AG, The Netherlands). Cyclic voltammetry (CV) measurements were carried out at room temperature in PBS. The electrode modification pro- cess was investigated by electrochemical impedance spectroscopy (EIS), which was performed in 5.0 mM Fe(CN)63–/4– solution containing 0.1 M KCl. The IR spectra were recorded in KBr tablets on an ALPHA FTIR spectrometer manufactured by BrukerOp- tikGmbH (Germany). SEM microphotographs and elemental analysis by en- ergy-dispersive X-ray spectroscopy (EDS) were obtained with AURIGA CrossBeam (Carl Zeiss NTS, Germany). 2.3. Synthesis of CuADCA and working electrode modification Scheme 1 represents the routes of CuADCA synthesis and GCE modification. Briefly, 0.33 mL of distilled water, 0.33 mL of ethyl alcohol, and 0.33 mL of DMF were added to a 1.5 mL Eppendorf tube. Next, the resulting mixture was divided into two equal parts of 0.5 mL. 0.54 mmol (0.0092 g) of CuCl2∙2H2O was dissolved in the first part, 0.02925 mmol (0.0034 g) ADCA was dissolved in the sec- ond part. After the dissolution of both components, the so- lutions were mixed and stirred for 10 minutes. Then 12.5 μL of triethylamine was added to the tube and stirred for 1.5 hours. The precipitated blue crystals were isolated by centrifugation and washed 3 times with water, ethyl alco- hol and DMF and dried in air at 60 °C for 3 hours. Then the 5 µL of suspension of 1 mg of CuADCA per 1 mL of water prepared with ultrasonication was drop casted on GCE and dried in air. The modified electrode was named CuADCA@GCE. 2.4. Procedure of electrocatalytic creatinine detec- tion using CuADCA@GCE as the electrocatalyst The analytical procedure included the following stages: 1. A cyclic voltammogram of CuADCA@GCE was regis- tered in the working solution (10 mL of PBS), and anodic peak current was denoted as I0. 2. 100 µL of standard creatinine solution in PBS (10 mM) was added to the working solution, and a cyclic voltammo- gram was registered. Anodic peak current was denoted as Ist. 3. 100 µL of analyzed creatinine solution in PBS (sample) was added to the working solution, and a cyclic voltammo- gram was registered. Anodic peak current was denoted as Is. 4. The concentration of creatinine in the sample (Cx) was calculated as in Equation 1: 𝑐𝑥 = 𝑐st 𝐼st 𝐼𝑥 (1) 3. Results and Discussion 3.1. Characterization of CuADCA The CuADCA synthesis methods used in the past involved mixing a Cu2+ salt with ADCA with further slow water evaporation during several days [14]. To avoid long syn- thesis time, we use TEA as a deprotonating agent, so the reaction time reduces by up to 2 hours. The synthesis of the CuADCA can be explained by each ADCA ligand con- necting to two Cu2+ ions metal nodes, forming the linear polymer-like structure [14]. https://doi.org/10.15826/chimtech.2023.10.2.01 https://doi.org/10.15826/chimtech.2023.10.2.01 Chimica Techno Acta 2023, vol. 10(2), No. 202310201 LETTER 3 of 6 DOI: 10.15826/chimtech.2023.10.2.01 Scheme 1 CuADCA synthesis and electrode modification. The morphology, composition, and electrochemical properties of CuADCA were determined by SEM, EDS, FTIR, EIS and CV. Figure 1 shows the SEM image of CuADCA. As can be clearly seen, CuADCA particles appear layered, which is consistent with the polymer-like structure. The dimensions of the flakes vary from 1 µm to 20 µm. Successful synthesis of CuADCA material is confirmed by EDS (Table 1) and FTIR (Figure 2): the spectra of CuADCA synthesized using tri- ethylamine are identical to the spectra in the literature [16]. The spectra of ADCA have a characteristic peak at 1698 cm–1 (dimer between two carboxylic groups of the ADCA) that shifts to 1597 cm–1 in CuADCA spectra, which shows the pseudo-monodentate coordination of the anion. Nyquist plots for bare GCE and CuADCA@GCE were reg- istered in the solution containing 5 mM K4[Fe(CN)6]/K3[Fe(CN)6] and 0.1 M KCl at a potential of 0.25 V (Figure 3). Compared with the small semicircle of GCE, the Rct of CuADCA@GCE was increased, indicating the successful modification of CuADCA nanoparticles on the GCE surface. The increased Rct is due to the poor conductiv- ity of CuADCA, which hinders, to some extent, the electron transfer through the electrode-solution interface. 3.2. Electrochemical behaviour of CuADCA in the presence of creatinine Figure 4a shows cyclic voltammograms of bare GCE and CuADCA@GCE registered in 0.1 M PBS pH = 7.4 in absence and in presence of different concentrations of creatinine. As seen, a couple of cathodic (at –0.4 V) and anodic (at –0.1 V) peaks in CuADCA@GCE are observed. To determine the number of electrons involved in the electrochemical reac- tion, Heyrovsky-Ilkovic equation (Equation 2) was used: 𝐸 = 𝐸1 2⁄ + 𝑅𝑇 𝑛𝐹 ln ( 𝐼d − 𝐼 𝐼 ), (2) where R is the gas constant; T is the absolute temperature, K; F is the Faraday constant; n denotes the number of elec- trons taking part in the electrode reaction; E1/2 is the half- wave potential, V; E is the potential applied, V; I is the cur- rent registered when E is applied, A; Id is the limiting diffu- sion current (peak current), A. After rebuilding of voltammo- grams in the ranges of [(–0.20 V)–0 V] and [(–0.26 V)–(–0.35 V)], the numbers of electrons involved in the electrochemical reaction were calculated as na = 1.3, nc = 0.4. Deviations from a whole number can be explained as processes of reduction of Cu2+ and Cu1+ to Cu0 and further ox- idation of Cu0 to Cu1+ occurring simultaneously. The presence of creatinine, urea, or glucose in the work- ing solution leads to the increase in the peaks currents, and the additional current depends linearly on the analyte con- centration (Figure 4a, for creatinine). This fact can be ex- plained as electrocatalytic processes represented by Equa- tions 3–5: Cu+2 + e− → Cu+1, (3) Cu+1 − e− → Cu+2, (4) Cu+2 + A → Cu+1 + OxA. (5) Copper in form of Cu2+ binds with the analyte molecule (A) and then Cu2+ oxidizes the analyte molecule, is converted to Cu+1 while oxidizing the analyte to form OxA, so CuADCA acts as a catalyst of electrochemical oxidation of analytes. Figure 1 SEM microphotograph of CuADCA. Green arrows point to layered structure of microparticles. Table 1 Elemental composition of CuADCA in atomic %, measured with EDS. Spectrum numbers correspond to points in Figure 1. Spectrum number C O Cl Cu Spectrum 1 54.3 11.6 2.7 31.3 Spectrum 2 57.7 18.4 4.2 19.7 https://doi.org/10.15826/chimtech.2023.10.2.01 https://doi.org/10.15826/chimtech.2023.10.2.01 Chimica Techno Acta 2023, vol. 10(2), No. 202310201 LETTER 4 of 6 DOI: 10.15826/chimtech.2023.10.2.01 Figure 2 FTIR spectra of CuCl2, ADCA and CuADCA. Figure 3 Nyquist plots at potential of 0.25 V for bare GCE and CuADCA@GCE registered in 5 mM K4[Fe(CN)6]/K3[Fe(CN)6] + 0.1 M KCl; dots are experimental data and lines are fitted data. In- set: equivalent circuit. Electrochemical transformations of CuADCA are process es that are controlled by diffusion of analyte to electrode- solution interface; the linear plots of the dependence of peak current on the square root of scan rate (Figure 4b) in- dicate this. The analytical characteristics of urea, glucose, and cre- atinine determination were calculated from I vs. CA depend- ences and are reflected in Table 2. As seen, the most linear dependences were obtained for the anodic peaks, and the highest sensitivity (the slope of I vs. CA dependence) and the lowest limit of detection (LOD) were obtained for cre- atinine, which can be explained by the formation of strong complex between copper ions and creatinine molecules. This fact can be used in the future in the development of enzymeless electrochemical sensor for detection of creati- nine in the presence of glucose and urea. The developed creatinine determination system of CuADCA@GCE was compared with known enzymeless cre- atinine sensors (Table 3). It can be noticed that CuADCA@GCE is not inferior in sensitivity and LOD to the previously developed sensors. Figure 4 Cyclic voltammograms of bare GCE and CuADCA@GCE in absence and in present of 10 µM, 20 µM, 30 µM and 40 µM of creati- nine in 0.1 M PBS pH = 7.4. Scan rate: 0.1 V/s (a). The dependence of anodic and cathodic current peaks on square root of scan rate of CuADCA@GCE in 0.1 M PBS pH = 7.4 with 40 µM of creatinine (b). 4. Limitations In this research, creatinine, urea and glucose were deter- mined in model solutions imitating blood serum. In order to develop the enzymeless electrochemical sensor for creat- inine, it is necessary to make investigations with real blood serum and whole blood. https://doi.org/10.15826/chimtech.2023.10.2.01 https://doi.org/10.15826/chimtech.2023.10.2.01 Chimica Techno Acta 2023, vol. 10(2), No. 202310201 LETTER 5 of 6 DOI: 10.15826/chimtech.2023.10.2.01 Table 2 Analytical characteristics of urea, glucose, and creatinine determination with CuADCA@GCE as electrocatalyst (n = 5, P = 0.95). Analyte Sensitivity (µA/mM) R2 LOD (µM) Linear range (mM) Anodic peak Cathodic peak Anodic peak Cathodic peak Urea 1.0±0.1 5.0±0.4 0.973 0.891 1923 0–10 Glucose 13.0±1.1 116±10 0.969 0.874 228 0–0.4 Creatinine 1057±99 1756±150 0.966 0.649 2 0–0.1 Table 3 Comparing of CuADCA@GCE with previously developed electrochemical enzymeless sensors for creatinine determination sys- tems. System Sensitivity, µA/mM LOD, µM Linear range, µM Reference CuADCA@GCE 1057±99 2 0–0.1 This work Fe2O3/PANI-1@GCE 28.77 0,23 0.442–8840 [17] CuO/IL@ERGO 1.02 0.2 0.01–2000 [18] CB nanoparticles@SPE 16.7 8.6 370–3600 [19] 5. Conclusions In this work we report enzymeless electrochemical deter- mination of urea, glucose, and creatinine in PBS pH = 7.4 on the glassy carbon electrode modified with CuADCA syn- thesized with a simple and rapid method and characterized by SEM, FTIR, EIS and CV. Sensitivity and LOD were calcu- lated. The results show that CuADCA immobilized on GCE acts as catalyst in electrooxidation reaction of glucose, urea, and creatinine. Since the sensitivity and LOD for cre- atinine detection were more than 100 times higher than for glucose and urea detection, CuADCA is a promising material for further development of an enzyme-free sensor for cre- atinine. As compared with the existing enzymeless sensors, CuADCA@GCE is not inferior in sensitivity and LOD. ● Supplementary materials No supplementary materials are available. ● Funding The research funding from the Ministry of Science and Higher Education of the Russian Federation (Ural Federal University project within the Priority-2030 Program) is gratefully acknowledged. ● Acknowledgments The equipment of the Ural Center for Shared Use “Modern nanotechnology” SNSM UrFU was used. ● Author contributions Conceptualization: A.V.O. Data curation: A.V.O. Formal Analysis: A.V.O., A.N.K. Funding acquisition: M.I.S., A.N.K. Investigation: A.A.I., Y.V.I. Methodology: A.V.O. Project administration: A.N.K. Resources: A.N.K., A.I.M. Software: A.V.O. Supervision: A.V.O. Validation: A.V.O., A.N.K. Visualization: M.I.S., Y.V.I. Writing – original draft: A.V.O. 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