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A novel pH sensor with application to milk based on electrochemical oxidative quinone-functionalization of tryptophan residues
Journal of Electroanalytical Chemistry 859 (2020) 113871
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A novel pH sensor with application to milk based on electrochemical oxidative quinone-functionalization of tryptophan residues ⁎
Gengxin Hu a, Nanxi Li a, Yuwei Zhang a, Hong Li a,b, a b
School of Chemistry, South China Normal University, Guangzhou 510006, PR China Engineering Research Center of MTEES, Ministry of Education, South China Normal University, Guangzhou 510006, PR China
A R T I C L E
I N F O
Article history: Received 25 November 2019 Received in revised form 8 January 2020 Accepted 19 January 2020 Available online 21 January 2020 Keywords: Tryptophan pH sensor Proton-coupled electron transfer Graphite electrode Protein
A B S T R A C T
The electrochemical oxidative quinone-functionalization of tryptophan (Trp) residues, and the proton-coupled electron transfer properties between quinonized Trp (Trp=O) and its re-reduced species (Trp-OH) are investigated for potentiometric pH sensors. The Trp=O/Trp-OH couples are assembled on a graphite electrode using multiple cyclic voltammetry, and exhibit a pair of well-defined redox peaks at redox potential of 0.090 V (vs. SCE) at pH 7.0 and 298 K. The proton-coupled electron transfer properties of Trp=O/Trp-OH couples are dependent on electrode substrate material, incubation temperature and pH values, and Trp residue sources including amino acid, peptide and protein. The Trp=O/Trp-OH-based surface-confined exothermic electrode reaction rates show a decrease with increasing incubation temperature from 283 to 323 K at pH 5.0. Both of redox peak potential and open-circuit potential under optimum conditions have a good linear pH dependence between 1.0 and 12.0 with sensitivity of more than 52 mV pH −1 and relative standard derivation of 1.5%. The proposed potentiometric pH sensor has significant advantages of low cost, good stability and reproducibility, and strong anti-interference ability. The present study shows a new approach to endow Trp, Trp-containing peptides and non-conjugated proteins with analogous proton-coupled electron transfer performance to prosthetic groups of quinoproteins for monitoring pH changes in milk samples. © 2020 Elsevier B.V. All rights reserved.
1. Introduction The pH measurement is essential importance in many fields, such as industry, agriculture, biology, medicine, and environmental monitoring [1,2]. The classical potentiometry to determine a solution pH value has been developed in the past decades [3–6]. However, pH sensing for food and healthcare applications usually requires some materials with good biocompatibility, and high sensitivity and anti-macrobiomolecule interference ability, and operational repeatability [7,8]. For example, because milk is regarded as a complete food, and often contains proteins of ca. 3% and lactose of approximately 4–5%, along with water and other necessary minerals and vitamins, its accurate pH detection needs to overcome the interference from proteins and sugars [9,10]. These potential interferences always limit effective applications of some potentiometric pH sensors in food and biological samples [11]. For this reason, various pH sensors have been investigated by introducing redox-active organic and inorganic compounds in the past decades [12,13]. Nevertheless, there is no report on proton-coupled electron transfer (PCET) properties of amino acid residues or their
⁎ Corresponding author at: School of Chemistry, South China Normal University, Guangzhou 510006, PR China. E-mail address: [email protected]. (H. Li).
http://dx.doi.org/10.1016/j.jelechem.2020.113871 1572-6657/© 2020 Elsevier B.V. All rights reserved.
quinonized products for potential applications to pH monitoring in dairy products. Some native proteins possess a wide variety of biological functions that are governed by PCET reactions with assistance of amino acid residues [14]. Tryptophan (Trp) or tyrosine (Tyr) is one of the most commonly oxidized or excited aromatic amino acid residues in proteins [15,16]. In general, the exposure of Trp residues in proteins to the outer aqueous environment or electrode surface may cause fluorescence quenching, or electrochemical oxidation [17,18]. Therefore, the Trpbased electrochemical oxidation and fluorescence spectroscopic methods have provided insight into protein unfolding/folding and PCET properties during enzyme- and non-enzyme-catalyzed reactions [19,20]. The pH-dependent oxidation of Trp residues was usually carried out with the aid of photo irradiation, external electric field, thermal motion, and chemical denaturants [21,22]. In comparison, the electrochemical techniques are more suitable to investigate Trp residuebased proton motion and electron transfer reactions, which are analogous to the dehydrogenase-catalyzed oxidation of organic reactants [23]. So far, much attention has been paid to direct electron transfer reactions of proteins using electrochemical methods in the past decades [24,25]. As we know, quinoproteins have prosthetic groups bearing quinones that play important roles in catalytic cycles, for which tryptophan tryptophylquinone (TTQ) and topaquinone/lysine tyrosylquinone
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Journal of Electroanalytical Chemistry 859 (2020) 113871
water for 20 min. Subsequently, the Trp=O/Trp-OH couples were assembled onto the Gr, GC or ITO substrates by repetitive cyclic voltammetry (Re-CVs) of 10 cycles in electrolyte I containing 1.0 mmol L−1 Trp, KFWGK or BSA in a potential range between −0.2 and 1.0 V (vs. SCE) at 0.10 V s −1 (Fig. 1). The resulting electrode was soaked in deionized water for 10 min to remove non-oxidized Trp, KFWGK or BSA. Unless otherwise noted, the experiments were conducted at room temperature (297–299 K).
(TPQ/LTQ) found in bacteria are derived from two Trp residues and Tyr together with a lysyl residue, respectively [26]. For non-conjugated proteins without non-protein components, overall, Trp and Tyr residues in peptides and proteins may be involved in an irreversible oxidation process at carbon electrodes [27,28]. However, Özcan and co-workers found that the Trp oxidation product, i.e., 2-amino-3-(5-oxo-3,5dihydro-2H-indol-3-yl)propionic acid (Trp=O) was reduced to 2amino-3-(5-hydroxy-3,5-dihydro-2H-indol-3-yl)propionic acid (TrpOH) [29]. In our previous studies [30], the Trp=O/Trp-OH couples showed well-defined redox peaks on carbon nanotubes (CNTs). In the current work, a great effort is devoted to the electrochemical oxidative quninone-functionalization of Trp residues on graphite electrode, and the PCET properties of as-prepared Trp=O/Trp-OH couples that are analogous to quinone-containing prosthetic groups. Another emphasis is placed on Trp=O/Trp-OH-based potentiometric pH sensors with strong anti-interference ability. To the best of our knowledge, this is the first example of using amino acid oxidation products as a basis of developing potentiometric pH sensors, in particular, for monitoring pH changes in dairy products.
3. Results and discussion 3.1. Electrochemical quinone-functionalization of Trp residues As a protein without non-protein units, BSA contains two Trp residues including Trp-134 and Trp-212. To compare electrochemical oxidative quinone-functionalization of free Trp and Trp residues in peptide and protein, repetitive cyclic voltammograms (Re-CVs) are shown in Fig. 1a and Fig. S1. It is seen that the free Trp and Trp residues in peptide and BSA show an oxidation peak I at Gr electrode on the first cyclic voltammograms in the potential range between 0.4 and 1.0 V. While increasing scan number to the 10th cycle, the oxidation peak I height continuously decreases and a pair of new redox peaks emerges between −0.2 and 0.4 V, suggesting the oxidation of Trp units and the formation of redox-active centers via strong adsorption of oxidation products [30]. Additionally, the free Trp exhibits the largest oxidation peak current (peaks I and II), while BSA has the smallest with peptide being in between, revealing that the amino acid residues around Trp may prevent the direct contact of Trp residues in peptide and BSA with Gr electrode surface. On the other hand, as seen by Fig. S2, the peak current intensities of Trp on Gr electrode are far larger than that on GC and ITO electrodes, suggesting that the Gr structure facilitates the sufficient contact of Trp units with Gr surface. While the as-prepared electrode is transferred to electrolyte I, a pair of well-defined redox peaks is observed (Fig. 2 and Fig. S3). The redox peak potential (Erpp) is estimated to be 0.09 V at 0.10 V s−1, which is taken as an average of oxidation peak potential (0.075 V) and reduction peak potential (−0.057 V). This value is in agreement with that of PQQ/ PQQH2 couple (PQQ = pyrrolo-quinoline quinone) that acted as a redox carrier [32]. The redox peak potential difference is 132 mV, redox peak current ratio is close to 1.0, and peak current linearly increases with increasing scan rate. All results suggest that the redox reactions are responsible for surface-confined electron transfer processes. Combined with the characterization of Trp oxidation products reported previously [29], the Trp is oxidized to form Trp=O, which is reduced into Trp-OH, representing a Trp=O/Trp-OH-based redox reactions (Fig. 1b). This proposition is further verified by fluorescence spectra of Trp and its oxidation products (Fig. 1c). It is can be seen that Trp exhibits an emission peak at 369 nm, and the oxidation of Trp at 1.2 V for 5 min to Trp=O allows the emission peak to produce a blue shift of 13 nm. While the oxidation product is reduced to Trp-OH at 0 V, the enhanced emission peak appears at 359 nm and subsequently the Trp-OH is re-oxidized to Trp=O, making the emission peak shifted to 356 nm. Additionally, as depicted by Fig. 1d, although the Trp=O/Trp-OH film may be too thin to be distinctly seen and sufficiently identified, the nitrogen/oxygen elements along with C element from EDS measurements further demonstrate the presence of the modified film. To throw light on the effects of Trp sources and electrode substrate materials on electrochemical properties of Trp=O/Trp-OH couples, cyclic voltammograms and electrochemical impedance spectra were measured (Fig. 2). While the free Trp is changed to Trp-containing peptide, the redox peak current densities (peak II) show a large decrease, and the redox peak potential has a positive shift of 47 mV. While being changed to BSA, the redox peak current becomes smaller. The result is consistent with the changes from direct electrochemical oxidation (peak I). On the other hand, the replacement of Gr substrate with GC or ITO electrode leads to an obvious decrease in the redox peak height, and an increase in the charge transfer resistance (Table S1). Compared with
2. Experimental section 2.1. Chemicals and materials Bovine serum albumin (BSA), L-tryptophan (Trp), L-tyrosine (Tyr), Llysine (Lys), L-arginine (Arg), L-cystine (Cys) and L-phenylalanine (Phe) were purchased from Qisheng Biotechnique Co., Guangzhou, China. The pentapeptide with the sequence of Lys-Phe-Trp-Gly-Lys (Lys: lysine, Phe: phenylalanine, Gly: glycine) abbreviated to KFWGK was synthesized and characterized by ChinaPeptides Co., Ltd., Shanghai, China. A phosphate buffer solution (PBS) consisting of 0.05 mol L−1 Na2HPO4/ 0.05 mol L−1 NaH2PO4 of pH 7.0 was employed as electrolyte I, and 0.05 mol L−1 NaCl as electrolyte II. Prior to voltammetric measurements, the fresh solution was prepared, and adjusted to a desired pH value with diluted HCl or NaOH. D-Glucose (Glu), D-sucrose (Suc) and D-lactose (Lac) from Aladdin Reagent Co., Ltd., Shanghai, China, and other reagents were used as received. A graphite (Gr) disk (ϕ = 2 mm) was employed as working electrode, titanium sheet as counter electrode, and saturated calomel electrode (SCE) as reference electrode. For comparison, the Gr electrode was replaced with a glassy carbon (GC) disk (ϕ = 3 mm) or indium-tin oxide (ITO, 10 Ω sq.−1) sheet (0.56 cm2) from Shenzhen Nanbo Co., China. The effective area of the working electrode was determined by cyclic voltammograms of 5 mmol L−1 [Fe(CN) 3−/4− redox probes in 0.1 mol L−1 KCl with diffusion coefficient of 6] 7.6 × 10−6 cm2 s −1 [31]. 2.2. Instruments and methods Voltammetric measurements were performed on CHI660E electrochemical working station (Shanghai, China) in a homemade cell. Electrochemical impendence spectroscopy (EIS) was performed on an Autolab PGSTAT 302 electrochemical system with FRA software packages (Ecochemie, The Netherlands) in the frequency range between 100 kHz and 0.1 Hz with an amplitude of 5 mV at open-circuit potentials. The pH values were measured with a PHS-3C digital pH meter (Shanghai Leici Co., China). The emission spectra were recorded on an F-4600 fluorescence spectrophotometer (Hitachi, Japan). The Zeiss Ultra55 field emission scanning electron microscope (SEM, Germany) and energy dispersive spectroscopy (EDS) were used to characterize surface morphology and elemental composition of the modified electrodes. 2.3. Preparation of Trp=O/Trp-OH modified electrodes The Gr, GC or ITO electrode was employed as the substrates for the preparation of the modified layers. The Gr or GC disk was first polished with 0.05 mm alumina slurry, and then was sonicated in deionized 2
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Journal of Electroanalytical Chemistry 859 (2020) 113871
Fig. 1. Schematic diagrams for illustrating preparation, characterization and redox reactions of Trp=O/Trp-OH couple as a basis of potentiometric pH sensors. (a) Repetitive cyclic voltammograms (Re-CVs) of 1.0 mmol L−1 Trp on graphite (Gr) at 0.1 V s−1. (b) Electrochemical oxidation of Trp and proton-coupled electron transfer (PCET) reactions between Trp=O and Trp-OH. (c) Fluorescence spectra of bare ITO and modified electrodes obtained by the inserted procedures. (d) SEM and EDS analysis of (Trp=O/Trp-OH)/Gr. (e) Eocp from Trp=O/Trp-OH/Gr vs. SCE as a function of pH.
ITO and non-graphitizing amorphous GC substrates, graphite has stable hexagonal lattice, considerable porosity and π orbitals that are overlapped to form three-dimensional networks. Consequently, the Gr substrate provides more suitable electron bond networks that facilitate the adsorption and binding of Trp and Trp=O/Trp-OH couples on Gr surface. Thus, as seen by Table S1, the charge transfer resistance on Gr electrode is far smaller than that on GC or ITO electrode, and the film resistance exhibits an opposite change. The result demonstrates that the larger amounts of Trp=O/Trp-OH couples are formed on the Gr surface, leading to an increase in the redox peak current and a decrease in the charge transfer resistance. Therefore, the free Trp and Gr substrates are chosen to investigate the Trp=O/Trp-OH-based PCET performance and pH sensing below.
3.2. Temperature-dependent redox reactions of Trp=O/Trp-OH couples Because both oxidized and reduced forms are strongly adsorbed on the Gr surface, the surface-confined redox reactions of Trp=O/Trp-OH couples are described by Laviron's Eq. (1) [33]. Combined with CVs of (Trp=O/ Trp-OH)/Gr electrode in electrolyte II at different scan rates (υ, V s−1) in Fig. S3, the reaction rate constant (kf, cm s−1) is obtained. Ep;a ¼ Erpp −
RT βnF
ln
RT k f − lnυ βnF
ð1Þ
where Ep,a represents oxidative peak potential in V at a certain incubation temperature (T, K), and β is the electron transfer coefficient. When the 3
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Journal of Electroanalytical Chemistry 859 (2020) 113871
Fig. 3. (a) Oxidation peak potential (Ep,a) vs. lnυ plots for (Trp=O/Trp-OH)/Gr electrode in electrolyte II of pH 5.0 at different incubation temperatures (T): (1) 283, (2) 293, (3) 303, (4) 313 and (5) 323 K. (b) ln(kf/T) and lnkf with 1/T. Note that kf represents electrode reaction rate constant in cm s−1. (c) Open circuit potential (Eocp) vs. pH plots in electrolyte II at different incubation temperatures.
Fig. 2. (a) Cyclic voltammograms (CVs) of bare Gr (1), and modified Gr electrodes with Trp=O/Trp-OH couple generated from oxidation of Trp (2), KFWGK (3) or BSA (4) in electrolyte I at 0.1 V s−1. (b) CVs and (c) electrochemical impedance spectra (EIS) of Trp=O/Trp-OH couple assembled on Gr (1), GC (2) or ITO (3) substrates at open-circuit potentials.
by increasing incubation temperature. Combined with Arrhenium Eq. (2) and Eyring-Polanyi Eq. (3) [35], lnkf and ln(kf/T) are plotted against 1/T, respectively.
incubation temperature increases to 323 K from 283 K by keeping all other parameters constant, according to Fig. 3a and Fig. S3, the kf values are verified to be between 1.17 and 0.24 cm s−1 at Erpp, indicating fast Trp-O/TrpOH-based redox reactions [34], for which the rate constants are depressed
Ea lnk f ¼ − þ lnA RT 4
ð2Þ
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Journal of Electroanalytical Chemistry 859 (2020) 113871
kf H# kB S# þ ln ¼− þ ln RT R T h
ð3Þ
where Ea and A represent activation energy and pre-exponential factor, R and T are gas constant and absolute temperature, respectively. ΔH# and ΔS# represent activation enthalpy and activation entropy while kB and h are Boltzmann's and Planck's constants, Referring to Fig. 3b, the ΔH# value is estimated to be −31.6 kJ mol−1, indicating an exothermic reaction process [36]. The Ea value is −29.2 kJ mol−1. In general, the electrode reaction rate is increased by increasing incubation temperature [37]. The Trp=/Trp-OH-based redox reactions are responsible for surface-confined electron transfer processes, in which the reaction proceeding relies on the adsorption and binding intensities of Trp and Trp=O/Trp-OH couple on Gr electrode. The negative activation energies are typically barrier-less reactions that are driven by adsorption energy [38]. As a result, the Trp= O/Trp-OH-based reaction rate shows a decrease with increasing incubation temperature, which weakens the binding interaction of Trp=O/Trp-OH with Gr electrode due to thermal motion, and still is fit for an Arrhenius expression with a negative value of activation energy. The result offers a powerful theoretical basis to find optimum conditions of pH sensing. 3.3. Trp=O/Trp-OH-based proton-transfer equilibria and pH sensor The enzyme-catalyzed electron transfer reactions in biologically relevant systems are often coupled to proton transfer processes [39]. According to the composition and structure of Trp=O and Trp-OH, they may have potential functions that act as hydrogen bonding-assisted electron-proton transfer agents [40]. In water that acts as proton acceptor and donor via OH– and H3O+, proton transfer is relatively fast. If assuming the proton transfer to be at equilibrium, the measured equilibrium potential, where it is equal to redox peak potential (Erpp), has a pH dependence (Eq. 4) while [Trp=O] = [Trp-OH] [32]. Erpp ¼ E °0 −
2:303mRT pH nF
ð4Þ
Herein, E°′ represents the formal potential that is independent on pH if activity coefficients of Trp=O and Trp-OH are not influenced by solution pH values. m and n are the number of protons and electrons exchanged, respectively. The Erpp value is suggested to be dependent on pH changes. As seen by Fig. 4a, the redox peak potential shows a negative shift with increasing pH value in the range between 1.0 and 12.0. Because too high or too low pH values may influence the stability and existing forms of Trp= O/Trp-OH couple [32], the pH measurement range is controlled between 1.0 and 12.0. As depicted by the calibration plot based on cyclic voltammograms in Fig. 4b, the Erpp vs. pH plot for Trp=O/Trp-OH couple exhibits a negative linear correlation. The regression equation is described as Erpp = 0.44–0.057pH (R2 = 0.999), and the relative standard deviation is 1.5%, in which the mean error is generated from the measurements of three times at 298 K. The pH sensitivity is 57 mV pH −1, which is close to the theoretical value (59 mV pH −1), indicating characteristics of a 2e− and 2H+ transfer process, in which both of m and n are equal to 2 (Fig. 1b). Because the Trp=O/Trp-OH-based proton transfers are at equilibrium, the pH dependence of open-circuit potential (Eocp) is in agreement with that of Erpp, and conforms to a linear relationship at different incubation temperatures (Figs. 1e and 3c). It can be seen that the obtained response slopes exhibit an increase with increasing incubation temperature. The influences are verified to reply on the Eq. (4). Therefore, unless otherwise noted, the incubation temperature is set at 298 K.
Fig. 4. (a) CVs of (Trp=O/Trp-OH)/Gr electrode in electrolyte II at 0.1 V s−1 with different pH values: (1) 1.0, (2) 5.0, (3) 9.0 and (4) 12.0. (b) Redox peak potential (Erpp) vs. pH plots. (c) Eocp as a function of pH from (Trp=O/Trp-OH)/Gr electrode in electrolyte II after storing for (1) 1st, (2) 2nd, (3) 4th, (4) 8th and (5) 16th day.
3.4. Selectivity and reproducibility of proposed pH sensor Trp, peptide (KFWGK), BSA or milk hardly influences the voltammetric behavior and redox peak potentials of (Trp=O/Trp-OH)/Gr electrode in electrolyte I. On the other hand, the pH sensing is not influenced by high concentration (0.1 mol L−1) of saccharides such as glucose, sucrose and lactose, and amino acids (1.0 mmol L−1) including Cys, Tyr, Phe, Arg, Lys, etc.
The selectivity of Trp=O/Trp-OH-modified Gr electrode is further evaluated. As depicted by Fig. 5a, it is interesting to note that the proposed pH electrode is able to prevent the interference from Trp and other amino acids, Trp-containing peptides and proteins. The addition of 1.0 mmol L−1 5
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Journal of Electroanalytical Chemistry 859 (2020) 113871
solutions such as Trp and Tyr (Fig. 5a and b). Of course, the strongly oxidizing species may chemically degrade the Trp=O and Trp-OH, and further interfere with the pH measurement. Additionally, as seen by Fig. 4c, the (Trp=O/Trp-OH)/Gr electrodes show good linear responses to pH with a relatively constant slope over 16 days, suggesting that the proposed pH sensor has high stability and reproducibility.
3.5. pH determination in real samples The (Trp=O/Trp-OH)/Gr electrode in comparison with commercial glass electrode were used to detect the pH values of water, milk and cola samples [41]. Each sample was determined for three times. As depicted in Fig. 5c, the pH values of distilled water, milk and cola samples determined by using (Trp=O/Trp-OH)/Gr electrode are highly consistent with that using glass electrode, demonstrating that the (Trp=O/Trp-OH)/Gr electrode has comparable pH sensing performance with glass electrode. The proposed potentiometric sensor can be used to monitor pH changes in water, cola and milk samples, showing high selectivity and antiinterference ability. Additionally, while water molecules act as proton acceptor and donor, the (Trp=O/Trp-OH)/Gr electrode has ability to determine pH changes in aqueous samples without externally added buffers or supporting electrolytes.
4. Conclusions The Trp=O/Trp-OH couple that are successfully assembled on Gr electrode using multiple voltammetry via electrochemical oxidative quinonefunctionalization of free Trp, exhibit better redox activities than that of oxidized Trp residues in peptide and BSA, and that on GC or ITO electrode. The Trp=O/Trp-OH-based redox reactions are responsible for surfaceconfined electron transfer processes, and therefore show decreasing reaction rate constants with increasing incubation temperature from 283 to 323 K, and present exothermic PCET properties with negative activation energy. The (Trp=O/Trp-OH)/Gr electrode indicates a well linear response to pH changes in the range from 1.0 to 12.0 with sensitivity of more than 52 mV pH −1 that is close to theoretical value. The proposed potentiometric pH sensor has important advantages of low cost, good stability and reproducibility and high anti-interference ability. The results provide effective methods to endow Trp, Trp-containing peptides and non-conjugated proteins with analogous PCET performance to prosthetic groups of quinoproteins for monitoring pH changes in milk samples.
CRediT authorship contribution statement Gengxin Hu:Investigation, Data curation, Writing - original draft. Nanxi Li:Conceptualization, Software.Yuwei Zhang:Methodology, Validation.Hong Li:Supervision, Writing - review & editing.
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.
Fig. 5. (a) CVs of (Trp=O/Trp-OH)/Gr electrode in electrolyte I in the absence (1) and presence of 1.0 mmol L−1 Trp, KFWGK or BSA, or milk sample with pure milk/water volume ratio of 3:1. (b) Selectivity of the proposed pH sensors in electrolyte I containing 0.1 mol L−1 Glu, Suc and Lac, or 1.0 mmol L−1 Cys, Tyr, Phe, Arg and Lys using (Trp=O/Trp-OH)/Gr electrode. (c) pH sensing of water, pure milk and cola samples using (Trp=O/Trp-OH)/Gr and glass electrodes.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21271075). Appendix A. Supplementary data
(Fig. 5b). The results display that the (Trp=O/Trp-COH)/Gr pH electrode has large capacity of resisting disturbance of saccharides, amino acids and proteins. The main reason is that the Trp=O/Trp-OH couple adsorbed strongly on Gr depress the electrochemical oxidation of some species in
Supplementary data to this article can be found online at https://doi. org/10.1016/j.jelechem.2020.113871. 6
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7
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Journal of Electroanalytical Chemistry journal homepage: www.elsevier.com/locate/jelechem
A novel pH sensor with application to milk based on electrochemical oxidative quinone-functionalization of tryptophan residues ⁎
Gengxin Hu a, Nanxi Li a, Yuwei Zhang a, Hong Li a,b, a b
School of Chemistry, South China Normal University, Guangzhou 510006, PR China Engineering Research Center of MTEES, Ministry of Education, South China Normal University, Guangzhou 510006, PR China
A R T I C L E
I N F O
Article history: Received 25 November 2019 Received in revised form 8 January 2020 Accepted 19 January 2020 Available online 21 January 2020 Keywords: Tryptophan pH sensor Proton-coupled electron transfer Graphite electrode Protein
A B S T R A C T
The electrochemical oxidative quinone-functionalization of tryptophan (Trp) residues, and the proton-coupled electron transfer properties between quinonized Trp (Trp=O) and its re-reduced species (Trp-OH) are investigated for potentiometric pH sensors. The Trp=O/Trp-OH couples are assembled on a graphite electrode using multiple cyclic voltammetry, and exhibit a pair of well-defined redox peaks at redox potential of 0.090 V (vs. SCE) at pH 7.0 and 298 K. The proton-coupled electron transfer properties of Trp=O/Trp-OH couples are dependent on electrode substrate material, incubation temperature and pH values, and Trp residue sources including amino acid, peptide and protein. The Trp=O/Trp-OH-based surface-confined exothermic electrode reaction rates show a decrease with increasing incubation temperature from 283 to 323 K at pH 5.0. Both of redox peak potential and open-circuit potential under optimum conditions have a good linear pH dependence between 1.0 and 12.0 with sensitivity of more than 52 mV pH −1 and relative standard derivation of 1.5%. The proposed potentiometric pH sensor has significant advantages of low cost, good stability and reproducibility, and strong anti-interference ability. The present study shows a new approach to endow Trp, Trp-containing peptides and non-conjugated proteins with analogous proton-coupled electron transfer performance to prosthetic groups of quinoproteins for monitoring pH changes in milk samples. © 2020 Elsevier B.V. All rights reserved.
1. Introduction The pH measurement is essential importance in many fields, such as industry, agriculture, biology, medicine, and environmental monitoring [1,2]. The classical potentiometry to determine a solution pH value has been developed in the past decades [3–6]. However, pH sensing for food and healthcare applications usually requires some materials with good biocompatibility, and high sensitivity and anti-macrobiomolecule interference ability, and operational repeatability [7,8]. For example, because milk is regarded as a complete food, and often contains proteins of ca. 3% and lactose of approximately 4–5%, along with water and other necessary minerals and vitamins, its accurate pH detection needs to overcome the interference from proteins and sugars [9,10]. These potential interferences always limit effective applications of some potentiometric pH sensors in food and biological samples [11]. For this reason, various pH sensors have been investigated by introducing redox-active organic and inorganic compounds in the past decades [12,13]. Nevertheless, there is no report on proton-coupled electron transfer (PCET) properties of amino acid residues or their
⁎ Corresponding author at: School of Chemistry, South China Normal University, Guangzhou 510006, PR China. E-mail address: [email protected]. (H. Li).
http://dx.doi.org/10.1016/j.jelechem.2020.113871 1572-6657/© 2020 Elsevier B.V. All rights reserved.
quinonized products for potential applications to pH monitoring in dairy products. Some native proteins possess a wide variety of biological functions that are governed by PCET reactions with assistance of amino acid residues [14]. Tryptophan (Trp) or tyrosine (Tyr) is one of the most commonly oxidized or excited aromatic amino acid residues in proteins [15,16]. In general, the exposure of Trp residues in proteins to the outer aqueous environment or electrode surface may cause fluorescence quenching, or electrochemical oxidation [17,18]. Therefore, the Trpbased electrochemical oxidation and fluorescence spectroscopic methods have provided insight into protein unfolding/folding and PCET properties during enzyme- and non-enzyme-catalyzed reactions [19,20]. The pH-dependent oxidation of Trp residues was usually carried out with the aid of photo irradiation, external electric field, thermal motion, and chemical denaturants [21,22]. In comparison, the electrochemical techniques are more suitable to investigate Trp residuebased proton motion and electron transfer reactions, which are analogous to the dehydrogenase-catalyzed oxidation of organic reactants [23]. So far, much attention has been paid to direct electron transfer reactions of proteins using electrochemical methods in the past decades [24,25]. As we know, quinoproteins have prosthetic groups bearing quinones that play important roles in catalytic cycles, for which tryptophan tryptophylquinone (TTQ) and topaquinone/lysine tyrosylquinone
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water for 20 min. Subsequently, the Trp=O/Trp-OH couples were assembled onto the Gr, GC or ITO substrates by repetitive cyclic voltammetry (Re-CVs) of 10 cycles in electrolyte I containing 1.0 mmol L−1 Trp, KFWGK or BSA in a potential range between −0.2 and 1.0 V (vs. SCE) at 0.10 V s −1 (Fig. 1). The resulting electrode was soaked in deionized water for 10 min to remove non-oxidized Trp, KFWGK or BSA. Unless otherwise noted, the experiments were conducted at room temperature (297–299 K).
(TPQ/LTQ) found in bacteria are derived from two Trp residues and Tyr together with a lysyl residue, respectively [26]. For non-conjugated proteins without non-protein components, overall, Trp and Tyr residues in peptides and proteins may be involved in an irreversible oxidation process at carbon electrodes [27,28]. However, Özcan and co-workers found that the Trp oxidation product, i.e., 2-amino-3-(5-oxo-3,5dihydro-2H-indol-3-yl)propionic acid (Trp=O) was reduced to 2amino-3-(5-hydroxy-3,5-dihydro-2H-indol-3-yl)propionic acid (TrpOH) [29]. In our previous studies [30], the Trp=O/Trp-OH couples showed well-defined redox peaks on carbon nanotubes (CNTs). In the current work, a great effort is devoted to the electrochemical oxidative quninone-functionalization of Trp residues on graphite electrode, and the PCET properties of as-prepared Trp=O/Trp-OH couples that are analogous to quinone-containing prosthetic groups. Another emphasis is placed on Trp=O/Trp-OH-based potentiometric pH sensors with strong anti-interference ability. To the best of our knowledge, this is the first example of using amino acid oxidation products as a basis of developing potentiometric pH sensors, in particular, for monitoring pH changes in dairy products.
3. Results and discussion 3.1. Electrochemical quinone-functionalization of Trp residues As a protein without non-protein units, BSA contains two Trp residues including Trp-134 and Trp-212. To compare electrochemical oxidative quinone-functionalization of free Trp and Trp residues in peptide and protein, repetitive cyclic voltammograms (Re-CVs) are shown in Fig. 1a and Fig. S1. It is seen that the free Trp and Trp residues in peptide and BSA show an oxidation peak I at Gr electrode on the first cyclic voltammograms in the potential range between 0.4 and 1.0 V. While increasing scan number to the 10th cycle, the oxidation peak I height continuously decreases and a pair of new redox peaks emerges between −0.2 and 0.4 V, suggesting the oxidation of Trp units and the formation of redox-active centers via strong adsorption of oxidation products [30]. Additionally, the free Trp exhibits the largest oxidation peak current (peaks I and II), while BSA has the smallest with peptide being in between, revealing that the amino acid residues around Trp may prevent the direct contact of Trp residues in peptide and BSA with Gr electrode surface. On the other hand, as seen by Fig. S2, the peak current intensities of Trp on Gr electrode are far larger than that on GC and ITO electrodes, suggesting that the Gr structure facilitates the sufficient contact of Trp units with Gr surface. While the as-prepared electrode is transferred to electrolyte I, a pair of well-defined redox peaks is observed (Fig. 2 and Fig. S3). The redox peak potential (Erpp) is estimated to be 0.09 V at 0.10 V s−1, which is taken as an average of oxidation peak potential (0.075 V) and reduction peak potential (−0.057 V). This value is in agreement with that of PQQ/ PQQH2 couple (PQQ = pyrrolo-quinoline quinone) that acted as a redox carrier [32]. The redox peak potential difference is 132 mV, redox peak current ratio is close to 1.0, and peak current linearly increases with increasing scan rate. All results suggest that the redox reactions are responsible for surface-confined electron transfer processes. Combined with the characterization of Trp oxidation products reported previously [29], the Trp is oxidized to form Trp=O, which is reduced into Trp-OH, representing a Trp=O/Trp-OH-based redox reactions (Fig. 1b). This proposition is further verified by fluorescence spectra of Trp and its oxidation products (Fig. 1c). It is can be seen that Trp exhibits an emission peak at 369 nm, and the oxidation of Trp at 1.2 V for 5 min to Trp=O allows the emission peak to produce a blue shift of 13 nm. While the oxidation product is reduced to Trp-OH at 0 V, the enhanced emission peak appears at 359 nm and subsequently the Trp-OH is re-oxidized to Trp=O, making the emission peak shifted to 356 nm. Additionally, as depicted by Fig. 1d, although the Trp=O/Trp-OH film may be too thin to be distinctly seen and sufficiently identified, the nitrogen/oxygen elements along with C element from EDS measurements further demonstrate the presence of the modified film. To throw light on the effects of Trp sources and electrode substrate materials on electrochemical properties of Trp=O/Trp-OH couples, cyclic voltammograms and electrochemical impedance spectra were measured (Fig. 2). While the free Trp is changed to Trp-containing peptide, the redox peak current densities (peak II) show a large decrease, and the redox peak potential has a positive shift of 47 mV. While being changed to BSA, the redox peak current becomes smaller. The result is consistent with the changes from direct electrochemical oxidation (peak I). On the other hand, the replacement of Gr substrate with GC or ITO electrode leads to an obvious decrease in the redox peak height, and an increase in the charge transfer resistance (Table S1). Compared with
2. Experimental section 2.1. Chemicals and materials Bovine serum albumin (BSA), L-tryptophan (Trp), L-tyrosine (Tyr), Llysine (Lys), L-arginine (Arg), L-cystine (Cys) and L-phenylalanine (Phe) were purchased from Qisheng Biotechnique Co., Guangzhou, China. The pentapeptide with the sequence of Lys-Phe-Trp-Gly-Lys (Lys: lysine, Phe: phenylalanine, Gly: glycine) abbreviated to KFWGK was synthesized and characterized by ChinaPeptides Co., Ltd., Shanghai, China. A phosphate buffer solution (PBS) consisting of 0.05 mol L−1 Na2HPO4/ 0.05 mol L−1 NaH2PO4 of pH 7.0 was employed as electrolyte I, and 0.05 mol L−1 NaCl as electrolyte II. Prior to voltammetric measurements, the fresh solution was prepared, and adjusted to a desired pH value with diluted HCl or NaOH. D-Glucose (Glu), D-sucrose (Suc) and D-lactose (Lac) from Aladdin Reagent Co., Ltd., Shanghai, China, and other reagents were used as received. A graphite (Gr) disk (ϕ = 2 mm) was employed as working electrode, titanium sheet as counter electrode, and saturated calomel electrode (SCE) as reference electrode. For comparison, the Gr electrode was replaced with a glassy carbon (GC) disk (ϕ = 3 mm) or indium-tin oxide (ITO, 10 Ω sq.−1) sheet (0.56 cm2) from Shenzhen Nanbo Co., China. The effective area of the working electrode was determined by cyclic voltammograms of 5 mmol L−1 [Fe(CN) 3−/4− redox probes in 0.1 mol L−1 KCl with diffusion coefficient of 6] 7.6 × 10−6 cm2 s −1 [31]. 2.2. Instruments and methods Voltammetric measurements were performed on CHI660E electrochemical working station (Shanghai, China) in a homemade cell. Electrochemical impendence spectroscopy (EIS) was performed on an Autolab PGSTAT 302 electrochemical system with FRA software packages (Ecochemie, The Netherlands) in the frequency range between 100 kHz and 0.1 Hz with an amplitude of 5 mV at open-circuit potentials. The pH values were measured with a PHS-3C digital pH meter (Shanghai Leici Co., China). The emission spectra were recorded on an F-4600 fluorescence spectrophotometer (Hitachi, Japan). The Zeiss Ultra55 field emission scanning electron microscope (SEM, Germany) and energy dispersive spectroscopy (EDS) were used to characterize surface morphology and elemental composition of the modified electrodes. 2.3. Preparation of Trp=O/Trp-OH modified electrodes The Gr, GC or ITO electrode was employed as the substrates for the preparation of the modified layers. The Gr or GC disk was first polished with 0.05 mm alumina slurry, and then was sonicated in deionized 2
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Journal of Electroanalytical Chemistry 859 (2020) 113871
Fig. 1. Schematic diagrams for illustrating preparation, characterization and redox reactions of Trp=O/Trp-OH couple as a basis of potentiometric pH sensors. (a) Repetitive cyclic voltammograms (Re-CVs) of 1.0 mmol L−1 Trp on graphite (Gr) at 0.1 V s−1. (b) Electrochemical oxidation of Trp and proton-coupled electron transfer (PCET) reactions between Trp=O and Trp-OH. (c) Fluorescence spectra of bare ITO and modified electrodes obtained by the inserted procedures. (d) SEM and EDS analysis of (Trp=O/Trp-OH)/Gr. (e) Eocp from Trp=O/Trp-OH/Gr vs. SCE as a function of pH.
ITO and non-graphitizing amorphous GC substrates, graphite has stable hexagonal lattice, considerable porosity and π orbitals that are overlapped to form three-dimensional networks. Consequently, the Gr substrate provides more suitable electron bond networks that facilitate the adsorption and binding of Trp and Trp=O/Trp-OH couples on Gr surface. Thus, as seen by Table S1, the charge transfer resistance on Gr electrode is far smaller than that on GC or ITO electrode, and the film resistance exhibits an opposite change. The result demonstrates that the larger amounts of Trp=O/Trp-OH couples are formed on the Gr surface, leading to an increase in the redox peak current and a decrease in the charge transfer resistance. Therefore, the free Trp and Gr substrates are chosen to investigate the Trp=O/Trp-OH-based PCET performance and pH sensing below.
3.2. Temperature-dependent redox reactions of Trp=O/Trp-OH couples Because both oxidized and reduced forms are strongly adsorbed on the Gr surface, the surface-confined redox reactions of Trp=O/Trp-OH couples are described by Laviron's Eq. (1) [33]. Combined with CVs of (Trp=O/ Trp-OH)/Gr electrode in electrolyte II at different scan rates (υ, V s−1) in Fig. S3, the reaction rate constant (kf, cm s−1) is obtained. Ep;a ¼ Erpp −
RT βnF
ln
RT k f − lnυ βnF
ð1Þ
where Ep,a represents oxidative peak potential in V at a certain incubation temperature (T, K), and β is the electron transfer coefficient. When the 3
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Fig. 3. (a) Oxidation peak potential (Ep,a) vs. lnυ plots for (Trp=O/Trp-OH)/Gr electrode in electrolyte II of pH 5.0 at different incubation temperatures (T): (1) 283, (2) 293, (3) 303, (4) 313 and (5) 323 K. (b) ln(kf/T) and lnkf with 1/T. Note that kf represents electrode reaction rate constant in cm s−1. (c) Open circuit potential (Eocp) vs. pH plots in electrolyte II at different incubation temperatures.
Fig. 2. (a) Cyclic voltammograms (CVs) of bare Gr (1), and modified Gr electrodes with Trp=O/Trp-OH couple generated from oxidation of Trp (2), KFWGK (3) or BSA (4) in electrolyte I at 0.1 V s−1. (b) CVs and (c) electrochemical impedance spectra (EIS) of Trp=O/Trp-OH couple assembled on Gr (1), GC (2) or ITO (3) substrates at open-circuit potentials.
by increasing incubation temperature. Combined with Arrhenium Eq. (2) and Eyring-Polanyi Eq. (3) [35], lnkf and ln(kf/T) are plotted against 1/T, respectively.
incubation temperature increases to 323 K from 283 K by keeping all other parameters constant, according to Fig. 3a and Fig. S3, the kf values are verified to be between 1.17 and 0.24 cm s−1 at Erpp, indicating fast Trp-O/TrpOH-based redox reactions [34], for which the rate constants are depressed
Ea lnk f ¼ − þ lnA RT 4
ð2Þ
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kf H# kB S# þ ln ¼− þ ln RT R T h
ð3Þ
where Ea and A represent activation energy and pre-exponential factor, R and T are gas constant and absolute temperature, respectively. ΔH# and ΔS# represent activation enthalpy and activation entropy while kB and h are Boltzmann's and Planck's constants, Referring to Fig. 3b, the ΔH# value is estimated to be −31.6 kJ mol−1, indicating an exothermic reaction process [36]. The Ea value is −29.2 kJ mol−1. In general, the electrode reaction rate is increased by increasing incubation temperature [37]. The Trp=/Trp-OH-based redox reactions are responsible for surface-confined electron transfer processes, in which the reaction proceeding relies on the adsorption and binding intensities of Trp and Trp=O/Trp-OH couple on Gr electrode. The negative activation energies are typically barrier-less reactions that are driven by adsorption energy [38]. As a result, the Trp= O/Trp-OH-based reaction rate shows a decrease with increasing incubation temperature, which weakens the binding interaction of Trp=O/Trp-OH with Gr electrode due to thermal motion, and still is fit for an Arrhenius expression with a negative value of activation energy. The result offers a powerful theoretical basis to find optimum conditions of pH sensing. 3.3. Trp=O/Trp-OH-based proton-transfer equilibria and pH sensor The enzyme-catalyzed electron transfer reactions in biologically relevant systems are often coupled to proton transfer processes [39]. According to the composition and structure of Trp=O and Trp-OH, they may have potential functions that act as hydrogen bonding-assisted electron-proton transfer agents [40]. In water that acts as proton acceptor and donor via OH– and H3O+, proton transfer is relatively fast. If assuming the proton transfer to be at equilibrium, the measured equilibrium potential, where it is equal to redox peak potential (Erpp), has a pH dependence (Eq. 4) while [Trp=O] = [Trp-OH] [32]. Erpp ¼ E °0 −
2:303mRT pH nF
ð4Þ
Herein, E°′ represents the formal potential that is independent on pH if activity coefficients of Trp=O and Trp-OH are not influenced by solution pH values. m and n are the number of protons and electrons exchanged, respectively. The Erpp value is suggested to be dependent on pH changes. As seen by Fig. 4a, the redox peak potential shows a negative shift with increasing pH value in the range between 1.0 and 12.0. Because too high or too low pH values may influence the stability and existing forms of Trp= O/Trp-OH couple [32], the pH measurement range is controlled between 1.0 and 12.0. As depicted by the calibration plot based on cyclic voltammograms in Fig. 4b, the Erpp vs. pH plot for Trp=O/Trp-OH couple exhibits a negative linear correlation. The regression equation is described as Erpp = 0.44–0.057pH (R2 = 0.999), and the relative standard deviation is 1.5%, in which the mean error is generated from the measurements of three times at 298 K. The pH sensitivity is 57 mV pH −1, which is close to the theoretical value (59 mV pH −1), indicating characteristics of a 2e− and 2H+ transfer process, in which both of m and n are equal to 2 (Fig. 1b). Because the Trp=O/Trp-OH-based proton transfers are at equilibrium, the pH dependence of open-circuit potential (Eocp) is in agreement with that of Erpp, and conforms to a linear relationship at different incubation temperatures (Figs. 1e and 3c). It can be seen that the obtained response slopes exhibit an increase with increasing incubation temperature. The influences are verified to reply on the Eq. (4). Therefore, unless otherwise noted, the incubation temperature is set at 298 K.
Fig. 4. (a) CVs of (Trp=O/Trp-OH)/Gr electrode in electrolyte II at 0.1 V s−1 with different pH values: (1) 1.0, (2) 5.0, (3) 9.0 and (4) 12.0. (b) Redox peak potential (Erpp) vs. pH plots. (c) Eocp as a function of pH from (Trp=O/Trp-OH)/Gr electrode in electrolyte II after storing for (1) 1st, (2) 2nd, (3) 4th, (4) 8th and (5) 16th day.
3.4. Selectivity and reproducibility of proposed pH sensor Trp, peptide (KFWGK), BSA or milk hardly influences the voltammetric behavior and redox peak potentials of (Trp=O/Trp-OH)/Gr electrode in electrolyte I. On the other hand, the pH sensing is not influenced by high concentration (0.1 mol L−1) of saccharides such as glucose, sucrose and lactose, and amino acids (1.0 mmol L−1) including Cys, Tyr, Phe, Arg, Lys, etc.
The selectivity of Trp=O/Trp-OH-modified Gr electrode is further evaluated. As depicted by Fig. 5a, it is interesting to note that the proposed pH electrode is able to prevent the interference from Trp and other amino acids, Trp-containing peptides and proteins. The addition of 1.0 mmol L−1 5
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Journal of Electroanalytical Chemistry 859 (2020) 113871
solutions such as Trp and Tyr (Fig. 5a and b). Of course, the strongly oxidizing species may chemically degrade the Trp=O and Trp-OH, and further interfere with the pH measurement. Additionally, as seen by Fig. 4c, the (Trp=O/Trp-OH)/Gr electrodes show good linear responses to pH with a relatively constant slope over 16 days, suggesting that the proposed pH sensor has high stability and reproducibility.
3.5. pH determination in real samples The (Trp=O/Trp-OH)/Gr electrode in comparison with commercial glass electrode were used to detect the pH values of water, milk and cola samples [41]. Each sample was determined for three times. As depicted in Fig. 5c, the pH values of distilled water, milk and cola samples determined by using (Trp=O/Trp-OH)/Gr electrode are highly consistent with that using glass electrode, demonstrating that the (Trp=O/Trp-OH)/Gr electrode has comparable pH sensing performance with glass electrode. The proposed potentiometric sensor can be used to monitor pH changes in water, cola and milk samples, showing high selectivity and antiinterference ability. Additionally, while water molecules act as proton acceptor and donor, the (Trp=O/Trp-OH)/Gr electrode has ability to determine pH changes in aqueous samples without externally added buffers or supporting electrolytes.
4. Conclusions The Trp=O/Trp-OH couple that are successfully assembled on Gr electrode using multiple voltammetry via electrochemical oxidative quinonefunctionalization of free Trp, exhibit better redox activities than that of oxidized Trp residues in peptide and BSA, and that on GC or ITO electrode. The Trp=O/Trp-OH-based redox reactions are responsible for surfaceconfined electron transfer processes, and therefore show decreasing reaction rate constants with increasing incubation temperature from 283 to 323 K, and present exothermic PCET properties with negative activation energy. The (Trp=O/Trp-OH)/Gr electrode indicates a well linear response to pH changes in the range from 1.0 to 12.0 with sensitivity of more than 52 mV pH −1 that is close to theoretical value. The proposed potentiometric pH sensor has important advantages of low cost, good stability and reproducibility and high anti-interference ability. The results provide effective methods to endow Trp, Trp-containing peptides and non-conjugated proteins with analogous PCET performance to prosthetic groups of quinoproteins for monitoring pH changes in milk samples.
CRediT authorship contribution statement Gengxin Hu:Investigation, Data curation, Writing - original draft. Nanxi Li:Conceptualization, Software.Yuwei Zhang:Methodology, Validation.Hong Li:Supervision, Writing - review & editing.
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.
Fig. 5. (a) CVs of (Trp=O/Trp-OH)/Gr electrode in electrolyte I in the absence (1) and presence of 1.0 mmol L−1 Trp, KFWGK or BSA, or milk sample with pure milk/water volume ratio of 3:1. (b) Selectivity of the proposed pH sensors in electrolyte I containing 0.1 mol L−1 Glu, Suc and Lac, or 1.0 mmol L−1 Cys, Tyr, Phe, Arg and Lys using (Trp=O/Trp-OH)/Gr electrode. (c) pH sensing of water, pure milk and cola samples using (Trp=O/Trp-OH)/Gr and glass electrodes.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 21271075). Appendix A. Supplementary data
(Fig. 5b). The results display that the (Trp=O/Trp-COH)/Gr pH electrode has large capacity of resisting disturbance of saccharides, amino acids and proteins. The main reason is that the Trp=O/Trp-OH couple adsorbed strongly on Gr depress the electrochemical oxidation of some species in
Supplementary data to this article can be found online at https://doi. org/10.1016/j.jelechem.2020.113871. 6
G. Hu et al.
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