Chemicals

Catechin (>=98%, TLC grade) was procured from Sigma (UK). A 0.1 M sodium phosphate buffer solution (pH 6.8) was prepared using disodium hydrogen phosphate, sodium dihydrogen phosphate and potassium chloride, all obtained from Merck (India). Milli-Q water (Millipore India Ltd., India) was used throughout for the preparation of buffer solutions. All other reagents employed in the study were of analytical grade and were purchased from Merck (India). Commercially available green and black tea samples were collected from local markets. For HPLC analysis, acetonitrile, acetic acid and water of HPLC grade were obtained from Merck (India).

Instrumentation

Electrochemical measurements, including amperometric responses, were carried out using an Autolab electrochemical analyser (PGSTAT 12, Ecochemie B.V., The Netherlands). The electrodes were connected to the corresponding terminals of the Autolab system through standard connectors. Experimental parameters such as scan rate, applied potential and measurement duration were controlled and recorded using General Purpose Electrochemical Software operated via a computer interfaced with the analyser.

Sensor Fabrication

The sensing system was designed and fabricated for the electrochemical detection of tea polyphenols using a disposable SPE platform. The SPE consisted of a carbon working electrode, a carbon counter electrode and an integrated silver/silver chloride reference electrode. These electrodes were screen-printed onto a 250 µm thick polyester substrate (Cadillac Plastic Ltd., Swindon, UK) using a DEK 248 screen-printing machine (DEK Printing Machines Ltd., Weymouth, UK).

The circular electrocatalytic working electrode was fabricated using a commercially available carbon powder (MCA Services Ltd., Cambridgeshire, UK), which was converted into a screen-printable paste by mixing with 2.5% (w/v) hydroxyethyl cellulose (HEC) in phosphate-buffered saline (PBS) at a weight ratio of 1:3. The reference electrode ink comprised silver paste containing 15% (w/w) silver chloride (MCA, UK) (Singleton et al., 1999). The counter electrode and conductive tracks connecting the electrodes to the measurement system were printed using I45R carbon ink (MCA, UK).

To electrically insulate the basal tracks from the measurement solution, an epoxy-based protective coating ink (242-SB, Agmet ESL Ltd., Reading, UK) was applied. Following fabrication, the electrodes were thermally cured at 125 °C for 2 h to ensure proper curing of the epoxy resin and to enhance the mechanical stability and electrochemical durability of the electrocatalytic surface, enabling prolonged use in aqueous media. The detailed configuration of the sensing system is illustrated in Figure 1.

Figure 1. Schematic Illustration of the Transducer Layout.

Note: All dimensions are given in mm.

Preparation of Tea Extract

A variety of commercially available green and black tea samples were procured from the local market. The green tea samples included Tetley, Darjeeling Longview, Twinings, Japanese green tea, Golden Tips and Lipton, while the black tea samples comprised CTC, Tulip Bari, BOPL and Twinings. These samples were assigned identification numbers from 1 to 10, respectively, and stored in airtight containers until further use.

For extract preparation, 0.1 g of each tea sample was accurately weighed and transferred to a beaker. Subsequently, 5 mL of freshly boiled distilled water was added, and the mixture was allowed to infuse for 5 min. The infusion was then filtered to remove insoluble residues, and the resulting supernatant was used directly as the sample for electrochemical measurements.

Electrochemical Measurement

Cyclic voltammetry was employed to investigate the electrochemical behaviour of catechin and to determine the optimal oxidation potential for subsequent amperometric measurements. The experiments were carried out using a 200 mg L^-1 catechin solution prepared in 0.1 M phosphate buffer solution (PBS, pH 6.8). The voltammograms were recorded over a potential window ranging from -1.0 V to +1.0 V at a scan rate of 0.001 V s^-1, with a step potential of 0.00305 V. For comparison, cyclic voltammetric measurements were also performed using a blank solution consisting of 5 mL of 0.1 M PBS mixed with 0.5 mL of reverse osmosis (RO) water under identical experimental conditions.

High-performance Liquid Chromatography Analysis

HPLC was employed as an independent and well-established analytical technique to validate the results obtained from the electrochemical detection of tea polyphenols. Catechin was used as the representative polyphenol standard for validation, owing to its predominance in tea and its well-characterised chromatographic behaviour. HPLC analysis was carried out using a Varian LC system (USA) comprising a ProStar 230 solvent delivery module coupled with a ProStar 330 photodiode array (PDA) detector. Chromatographic separation of phenolic compounds was achieved on an OmniSpher C18 reversed-phase column (25 cm ´ 4.6 mm, Varian, USA), which was protected by a ChromSep guard column (1 cm ´ 3 mm, Varian, USA). The use of a PDA detector enabled reliable detection and quantification of catechin based on its characteristic UV-visible absorbance, thereby providing a robust reference method for comparison with electrochemical sensor data.

For HPLC analysis, an accurately weighed 0.1 g of each tea sample was infused with 5 mL of freshly boiled distilled water in a boiling water bath for 5 min. The infusion was allowed to cool to room temperature and then filtered through filter paper to remove suspended solids.

The filtrate was subjected to a decaffeination step by liquid-liquid extraction with an equal volume of chloroform. After phase separation, the lower chloroform layer was carefully discarded. This extraction procedure was repeated twice to ensure efficient removal of caffeine, following the reported method (Copeland et al., 1997).

The decaffeinated aqueous phase was subsequently extracted with ethyl acetate. The ethyl acetate layer was collected in Eppendorf tubes and evaporated overnight at 45 °C to remove the solvent. The resulting dried residue was reconstituted in HPLC-grade water and injected into the HPLC system for analysis. Chromatographic separation and quantification were performed according to a previously reported method (Liang et al., 2001).

Electrochemical Study

The cyclic voltammograms of catechin exhibited a well-defined anodic peak, confirming the electroactive nature of catechin at the fabricated sensor surface. The anodic oxidation peak was observed at approximately +0.450 V (vs Ag/AgCl), corresponding to the electrochemical oxidation of phenolic hydroxyl groups present in catechin molecules (Figure 2).

Figure 2. Reaction Mechanism of the Oxidation of Phenolic Hydroxyl Groups Present in Catechin.

This potential was identified as the optimum oxidation potential, as represented in Figure 3, and was subsequently selected as the working potential for amperometric measurements.

Figure 3. Cyclic Voltammogram of the Developed Sensor for Catechin (200 mg L^-1).

To further evaluate the electrochemical response of the sensor towards varying catechin concentrations, amperometric responses were recorded for catechin solutions in the concentration range of 0-400 mg L^-1 under the same experimental conditions. As shown in Figure 4, the anodic peak current increased progressively with increasing catechin concentration, indicating a concentration-dependent electrochemical response. The well-defined and reproducible oxidation peaks demonstrate efficient electron transfer at the electrode interface and confirm the suitability of the fabricated sensor for quantitative determination of catechin and related tea polyphenols.

Figure 4. Calibration Curve for the Developed Sensor Using Different Catechin Concentrations.

Validation and Statistical Analysis

To validate the quantification of catechin (total polyphenols) in tea samples, the analytical performance of the proposed electrochemical sensor was evaluated by comparing it with the reference HPLC method. The results obtained from both techniques exhibited a strong linear relationship, with a coefficient of determination (R²) of 0.9877 and a correlation coefficient of 0.994, indicating excellent agreement between the two methods. The sensor demonstrated an LOD of 1.151 mg L^-1 and a LOQ of 3.838 mg L^-1. The catechin concentrations determined using HPLC closely matched those measured using the electrochemical platform, confirming the reliability and accuracy of the proposed sensor. The high correlation coefficient, approaching unity, further substantiates the strong agreement between the electrochemical and HPLC results, as illustrated in Figure 5A.

The repeatability of the electrochemical measurements was assessed by calculating the coefficient of variation (CV) for catechin determination in ten different tea samples. Each sample was analysed in triplicate using the fabricated sensor. As shown in Figure 5B, the CV values are <3% across all samples, demonstrating excellent precision and good repeatability of the proposed electrochemical sensing platform.

Figure 5. (A) Comparison of Tea Polyphenol Measurements in Our Device with the Standard Results Obtained from High-performance Liquid Chromatography (HPLC) for 10 Different Tea Samples that are Different from the Ones Used to Construct the Calibration Curve. The Experimental Data with Tea Polyphenol Concentrations Demonstrate Good Agreement Between the Present Method and the HPLC Method, with a Correlation Coefficient of 0.994. (B) Coefficient of Variation (CV) of Measured Data of Tea Polyphenols for 10 Samples Using the Present Device. The CV for Every Sample is <3%, Indicating Good Precision Among the Measurements.