Electrochemical Detection of Caffeic Acid on Diethyl 3,4-Dihydroxythiophene-2,5-Dicarboxylate-Modified Carbon Paste Electrode: Insights from Computational Analysis

Abstract

This study presents the electrochemical detection of caffeic acid using an ester (Diethyl 3,4-dihydroxythiophene-2,5-dicarboxylate)-modified carbon paste electrode (EMCPE). Caffeic acid, a naturally occurring hydroxycinnamic acid with antioxidant properties, was investigated due to its significance in food products and its potential health benefits. The modified electrode demonstrated enhanced sensitivity and selectivity for caffeic acid detection. Voltammetric methods were applied to evaluate the electrode performance. Results indicated that EMCPE has improved electron transfer kinetics and a lower detection limit compared unmodified electrode. Detection and quantification thresholds (LOD and LOQ) were found to be $3.12\times {10}^{-6}$ M and $1.04\times {10}^{-3}$ M. Density functional theory used to understand the electron transfer properties of Diethyl 3,4-dihydroxythiophene-2,5-dicarboxylate. The study highlights the potential of EMCPE as a reliable and cost-effective sensor to quantify caffeic acid across different sample matrices.

1. Introduction

Common secondary metabolites in the plant kingdom, diverse small molecule phenolics can be found in a various plant-based foods—ranging from fruits like apples, grapes, and citrus to vegetables such as tomatoes, broccoli, and leafy greens—along with cereals, teas, and fermented beverages like wine, are rich sources of phenolic compounds [1]. They can be electrochemically oxidized rather quickly while producing suitable quinoid forms, as indicated by their antioxidant properties [2]. All plant species produce 3,4-dihydroxycinnamic ccid, also referred to as caffeic acid (CA), a naturally occurring hydroxycinnamic acid, through the shikimic/phenylpropanoid pathway, which deamines phenylalanine to cinnamic acid, which is subsequently converted to caffeic acid (CA).

CA is a naturally occurring compound synthesized across plants via shikimic acid phenylpropanoid metabolic pathway [3]. This process deamines phenylalanine to cinnamic acid, which is then converted to caffeic acid and its byproducts [3].

Caffeic acid (CA) can be found in various dietary sources such as red wine, spices like cloves and star anise, coffee, olive oil, cruciferous vegetables including cabbage and kale, and fruits like strawberries and grapes [4]. CA is recognized as for its antioxidant potential and extensively researched in biochemistry biochemistry and medicine due to its biological activities, including anti-inflammatory [4], anticancer, antipruritic, anticarcinogenic [5], and immune regulation properties. These aspects have encouraged researchers to focus on the electrochemical detection of CA [6].

Several instrumental methods exist for detecting caffeic acid, including capillary gas chromatography [7], liquid chromatography [8], electrophoresis [9], spectrophotometry, and voltammetry [10]. Electrochemical sensor offer an efficient, selective, and economical method for accurately determining caffeic acid levels [11,12] for caffeic acid detection, focusing on chemically modified electrodes.

Electrochemical sensors are powerful tools for analyzing food products, particularly food antioxidants [10,11,12,13]. Their ability to be easily modified for high selectivity and sensitivity makes them advantageous over other analytical strategies in detecting electrochemically active materials.

Caffeic acid undergoes a reversible redox transformation, as shown in Figure 1 [14]. Additionally, caffeic acid is widely studied for its antioxidant and anti-inflammatory properties, with its redox cycling contributing to these effects [15,16].

In this work, we have employed a carbon paste electrode (CPE), which was modified using a laboratory-synthesized ester—diethyl 3,4-dihydroxythiophene-2,5-dicarboxylate. The resulting ester-modified carbon paste electrode (EMCPE) demonstrated significantly improved electrochemical performance in the detection of caffeic acid compared to the unmodified CPE.

In recent years, our research has focused on understanding the catalytic behavior of modified CPE through density functional theory (DFT)-based modeling [17,18,19]. To rationalize the enhanced sensing performance of EMCPE, we utilized DFT calculations to investigate the local electron transfer properties of the synthesized ester. These computational insights provide a theoretical basis for how the ester moiety contributes to the electrocatalytic activity of the modified electrode.

2. Experimentation

2.1. Synthesize of Diethyl 3,4-Dihydroxythiophene-2,5-Dicarboxylate

The ester Diethyl 3,4-dihydroxythiophene-2,5-dicarboxylate was synthesized by esterification of thiodiglycolic acid using ethyl alcohol and catalytic amount of sulphuric acid. Reflux the reaction mixture overnight to ensure complete conversion. The ester was solvent extracted using diethyl ether and dried with sodium sulfate according to the literature report [20]. Thidiglycolic acid was acquired from spectrochem and utilised exactly as supplied.

2.2. Preparation of Bare and Ester-Modified CPE

BCPE was fabricated by thoroughly hand-mixing graphite powder with silicone oil in a 70:30 ratio using a mortar and pestle for half an hour. Similarly, ester-modified carbon paste electrodes were obtained by blending the pre-formed BCPE with varying amounts of ester oil.

The electrochemical sensing performance of the electrodes was analyzed using a PalmSens Sensit Smart potentiostat (PalmSens BV, Houten, The Netherlands). The carbon paste electrodes were inserted into the cavity and used as the working electrode. All reagents, such as caffeic acid and phosphate buffer tablets, were obtained from Sigma-Aldrich, Bangalore, India.

2.3. Computational Methods DFT Studies

Density functional theory (DFT) applied in the deMon2K algorithm [21] with PBE correlation functionals [22] and TZVP basis sets [23] was used for optimization after the molecular structure of Ester models was displayed using the Sinapsis tool [24], as illustrated in Figure 2 geometry. Using an analytical method implemented in the deMon2K code, the Fulkui function [25,26] was computed for every species.

Using the energy of fkucukkolbrontier orbitals (${ϵ}_{\mathrm{HOMO}}$ and ${ϵ}_{\mathrm{LUMO}}$), the following formula was utilized to approximate the reactivity descriptors [27]: hardness, softness [28], ionization potential(IP), and electron affinity (EA).

$$\begin{array}{cc}\hfill \mathrm{IP}& \approx -{ϵ}_{\mathrm{HOMO}}\hfill \end{array}$$

(1)

$$\begin{array}{cc}\hfill \mathrm{EA}& \approx -{ϵ}_{\mathrm{LUMO}}\hfill \end{array}$$

(2)

$$\begin{array}{cc}\hfill \eta & ={\displaystyle \frac{1}{2}}{\left({\displaystyle \frac{\partial \mu }{\partial N}}\right)}_{\nu }={\displaystyle \frac{1}{2}}{\left({\displaystyle \frac{{\partial }^{2}E}{\partial N^2}}\right)}_{\nu }={\displaystyle \frac{1}{2}}(\mathrm{IP}-\mathrm{EA})\hfill \end{array}$$

(3)

$$\begin{array}{cc}\hfill S& ={\displaystyle \frac{1}{2\eta }}\hfill \end{array}$$

(4)

The Fukui function can be defined according to Equation (5) and calculated analytically [26,29].

$$f^{\pm }\left(r\right)\equiv \underset{\Delta N\to 0^{\pm }}{lim}{\displaystyle \frac{{\rho }^{N+\Delta N}\left(r\right)-{\rho }^N\left(r\right)}{\Delta N}}$$

(5)

where $\rho \left(r\right)$ is the electron density, N is the number of electrons in the system, and + and − signs correspond to addition or removal of electrons, respectively.

3. Results

3.1. Surface Morphology of BCPE and Ester-Modified CPE

The surface morphologies of the BCPE and EMCPE were characterized using FESEM, as shown in Figure 3. The BCPE (Figure 3a) exhibits a relatively smooth texture with densely arranged graphite particles, indicative of limited porosity and restricted surface accessibility. In contrast, the EMCPE (Figure 3b) reveals slight textural irregularities, likely induced by ester functionalization.

Though the morphological distinction is subtle, the presence of microstructural roughness in EMCPE may introduce marginally increased active surface area and improved analyte diffusion. These features, albeit minor, can influence electrochemical behavior by enhancing electron transfer kinetics and sensitivity.

Thus, despite the overall similarity in morphology, the chemical modification in EMCPE appears to confer electrochemical benefits not readily apparent from imaging alone, supporting its superior analytical performance.

3.2. Cyclic Voltammetric Behavior of $K_4$[Fe(CN)₆] on Bare and Ester-Modified CPE

Cyclic voltammetry (CV) using ${\mathrm{K}}_4$[Fe(CN)₆] as a redox marker was employed to analyze the electroactive surface areas of both the BCPE and EMCPE, as shown in Figure 4. Curves (a) does not displayed any voltammetric peak and (b) exhibit both oxidation and reduction peaks, demonstrating the redox activity of the system. The redox peak currents at the EMCPE are higher compared to the BCPE, indicating that the ester modification enhances the electrochemically active sites on the carbon surface. This suggests that the electrode modification plays a crucial role in enhancing electron transfer.

The surface area of the BCPE and EMCPE was determined using the Randles–Sevcik equation [31]:

$$I_p=2.69\times {10}^{5}A{D}^{1/2}{n}^{3/2}{\nu }^{1/2}C$$

(6)

where A refers to the electrode’s active area (${\mathrm{c}\mathrm{m}}^2$), n is the number of electrons transferred during the redox reaction, C is the concentration in mol/${\mathrm{c}\mathrm{m}}^3$, D is the diffusion coefficient, and $\nu$ represents the scan rate (V/s). The calculated surface areas of the BCPE and EMCPE were found to be 0.01029 ${\mathrm{c}\mathrm{m}}^2$ and 0.02346 ${\mathrm{c}\mathrm{m}}^2$, respectively. This indicates that the EMCPE’s active surface area is approximately 2.28 times larger than that of the BCPE. An increase in electrode surface area typically enhances its electrocatalytic activity, and these findings clearly demonstrate that the EMCPE significantly improves the current response of ${\mathrm{K}}_4$[Fe(CN)₆].

3.3. Computational Studies

3.3.1. Global Electron Transfer Properties

Diethyl 3,4-dihydroxythiophene-2,5-dicarboxylate’s computed electronic characteristics were utilised to estimate important reactivity descriptors. The values of the HOMO and LUMO energies were determined to be ${ϵ}_{\mathrm{HOMO}}=-5.535$ eV and ${ϵ}_{\mathrm{LUMO}}=-2.155$ eV, respectively. The ionisation potential (IP), electron affinity (EA), hardness ($\eta$), and softness (S) were calculated using these values: $\mathrm{IP}=5.535$ eV, $\mathrm{EA}=2.155$ eV, $\eta =1.690$ eV, and $S=0.296$ e${\mathrm{V}}^{-1}$. According to these values, diethyl 3,4-dihydroxythiophene-2,5-dicarboxylate has a moderate level of electronic softness, which may improve the kinetics of electron transfer when applied as a carbon paste electrode modifier for caffeic acid detection. Good charge transfer capability is implied by the comparatively small HOMO-LUMO gap, which is essential for electrocatalytic applications.

3.3.2. Local Electron Transfer Properties

Analytical Fukui function analysis is a powerful quantum chemical approach that helps in understanding the electron transfer mechanisms of electrode modifiers at the molecular level [32,33]. This method provides spatial insights into regions of electrophilic and nucleophilic activity, crucial for rationalizing redox behavior in electrochemical sensing.

The local electron transfer characteristics of Diethyl 3,4-dihydroxythiophene-2,5-dicarboxylate were investigated using frontier molecular orbitals and Fukui function analysis, which showed a clear division between electron-rich and electron-poor regions. Due to its high polarisability and the presence of lone pair electrons that aid in electron delocalisation, the sulphur atom is the primary location for the HOMO as shown in Figure 5a. This increases the sulphur atom’s ability to donate electrons, which makes it a good place for oxidation. In contrast, because of the strong electron-withdrawing nature of the ester functionalities, the LUMO is primarily located on the ethyl ester groups as shown in Figure 5b. These groups are vulnerable to reduction because they produce an electron-deficient area. These findings are corroborated by the Fukui function analysis, which shows that the ${\mathrm{f}}^{-}\left(\mathbf{r}\right)$ function, which indicates nucleophilic reactivity, is concentrated on the sulphur atom as shown in Figure 5c and that the ${\mathrm{f}}^{+}\left(\mathbf{r}\right)$ function, which indicates electrophilic reactivity, is localised around the ethyl ester groups as shown in Figure 5d. This distinct electronic distribution indicates that while reduction is anticipated at the ester moieties, oxidation is most likely to take place at the sulphur site. Diethyl 3,4-dihydroxythiophene-2,5-dicarboxylate is a promising modifier for carbon paste electrodes because of its well-defined redox behaviour, especially in electrochemical sensing applications like caffeic acid detection.

3.4. Electrochemical Behavior of Caffeic Acid on Bare and Ester-Modified CPE

The electrochemical behavior was evaluated in 0.1 M KCl containing $2.273\times {10}^{-4}$ M CA using both EMCPE and BCPE, with measurements conducted at a scan rate of 0.05 V/s, as illustrated in Figure 6. Cyclic voltammetry was employed to compare responses in the absence and presence of CA. No oxidation peak was observed in the blank solution (curve a), whereas distinct anodic responses appeared for CA at both BCPE (curve b) and EMCPE (curve c). The EMCPE delivered significantly amplified redox signals relative to the unmodified electrode, evidencing improved electron transfer characteristics. This enhancement confirms the positive influence of ester functionalization on the electrode surface, resulting in superior sensitivity for CA detection.

3.5. Influence of Scan Rate

The influence of scan rate on the redox behavior of caffeic acid (CA) was evaluated using cyclic voltammetry at the EMCPE in 0.1 mM CA solution, as depicted in Figure 7. The voltammograms recorded at scan rates ranging from 0.05 to 0.1 V/s revealed a linear relationship between the anodic peak current ($I_{pa}$) and the square root of the scan rate ($\sqrt{\nu }$), as shown in Figure 7b, with a correlation coefficient of $R^2=0.9996$. This indicates a diffusion-controlled and quasi-reversible electron transfer mechanism. A slight positive shift in the oxidation potential was observed with increasing scan rate, suggesting kinetic limitations in the electron transfer process at the EMCPE surface.

4. Effect of Concentration for Caffeic Acid

Figure 8a shows the modified electrode’s differential pulse voltammograms in 0.1 M KCl with CA. With increasing CA concentration, the oxidation peak current ($I_{pa}$) increased from $2.273\times {10}^{-4}$ M to $1.25\times {10}^{-3}$ M. Figure 8b. displays the regression calibration plot created from Ipa vs. Conc. of CA, with the estimated correlation coefficient ($R^2$ = 0.9997). The detection limit, quantification limit, and linear range were estimated using the standard equations [34,35]

$$\mathrm{LOD}={\displaystyle \frac{3\sigma }{\mathrm{slope}}},\mathrm{LOQ}={\displaystyle \frac{10\sigma }{\mathrm{slope}}}$$

(7)

Here $\sigma$ is the standard deviation of the blank signal. Thus, $3.12\times {10}^{-6}$ M is the LOD $1.04\times {10}^{-3}$ is LOQ, while $1.04\times {10}^{-3}$–$3.0\times {10}^{-4}$ M is the linear range.

Our redesigned electrode shows improved sensitivity when compared to certain previously reported sensors. For instance, Polymer modified electrode showed a limit of detection (LOD) of 3.91 $\mathsf{\mu }$M [36] for caffeic acid, whereas an Au modified electrode displayed detection limit of 4.24 $\mathsf{\mu }$M [37]. Our sensor, on the other hand, has a reduced limit of detection (LOD) of 3.12 $\mathsf{\mu }$M, demonstrating its improved capacity to detect minuscule amounts of caffeic acid. The synergistic interaction between the carbon paste matrix and the ester modifier, which facilitates effective electron transfer and analyte adsorption, may be the cause of this enhanced detection capabilities.

5. Repeatability, Reproducibility, and Stability Assessment

Repeatability is one of the most significant aspects that affects the accurate computation of CA. Under ideal conditions, CVs evaluate the reproducibility of the electrochemical sensor. EMCPE was used to identify CA solutions five times in a row. Between the subsequent readings, there was virtually little change in the peak current. The relative standard deviations (RSDs) of 4.9% showed that the improved electrode’s electrochemical reactions to CA were highly repeatable.

Meanwhile, the endurance of the electrochemical sensor has to be taken into account. Three parallel electrodes were fabricated to determine the binary mixture of CA (2.5 µM) in order to investigate the repeatability of EMCPE. The results indicate a RSD of 0.854%.

Additionally, the longevity of EMCPE was analysed over a month. At this point, ten tests were conducted concurrently. The modified electrodes $I_{pa}$ values of CA stayed high at 96.3% after 30 days at ambient temperature. EMCPE also demonstrated excellent operational stability. Additionally, the repeatability of the sensor was confirmed by testing five independently fabricated electrodes under identical conditions for sensing CA. The resulting RSDs were 4.6% and 5.1%, respectively, indicating a high level of reproducibility and consistency of EMCPE.

To further assess the operational lifetime of the electrode, its performance was evaluated under repeated electrochemical cycling. It was observed that the detection ability of the electrode began to diminish after 30 continuous scans, suggesting this as the practical limit for reliable sensing performance under successive use.

6. Real Sample Analysis

The practical viability of EMCPE for the determination of CA is performed on food samples, such as coffee decoction, by employing the CV method. The food samples were acquired from nearby supermarkets. Recovery analysis of CA detection in a decoction of coffee was analyzed by the standard addition method. The steps procedure followed for preparation of real sample are as follows: 3 mL of coffee dection sample without any pretreatment were diluted to 10 mL with a 0.1 M KCl solution. Different volumes say 150–250 $\mathsf{\mu }$M of this solution were mixed with a known concentration of CA. CV evaluated the prepared solution utilizing the EMCPE.

Table 1. Results of CA analysis coffee decoction sample.

SI/No.	CA Spiked ($\mathbf{\mu }$M)	CA Sensed ($\mathbf{\mu }$M)	Deviation ($\mathbf{\mu }$M)	Recovery (%)
1	150	144	6	96.0
2	200	196	4	98.0
3	250	246	4	98.4

presents the results obtained from the recovery studies. The recovery values and relative standard deviation (RSD) fall within a range that highlights the exceptional performance of the ester-modified carbon paste electrode (EMCPE). These results indicate that the developed sensor is suitable for detecting caffeic acid (CA) in coffee decoction. Data acquired using the differential pulse voltammetry (DPV) technique show recovery rates ranging from 96.0% to 98.4%, as summarized in Table 1. These results confirm that the fabricated electrode is highly effective for analyzing coffee decoction, demonstrating its reliability and suitability for practical applications.

7. Conclusions

This study presents the development of an voltammetric sensor based on an EMCPE for the precise detection of CA using CV. SEM analysis confirmed the successful modification of the electrode surface, enhancing its electrochemical properties. The EMCPE demonstrated superior analytical performance compared to conventional electrodes, exhibiting improved LOD, and a broader dynamic range for CA detection. Key parameters such as pH and electrode surface characteristics were systematically optimized to maximize the electrocatalytic response for CA oxidation. The sensor achieved a low detection limit and exhibited excellent linearity across a wide concentration range, making it effective for detecting CA in real-world samples, including coffee decoction. Overall, the proposed sensor offers a robust platform for the quantitative determination of CA in diverse analytical and clinical settings.