Development of a point-of-care testing sensor using polypyrrole/TiO₂ molecular imprinting technology for cinchocaine determination

2.1 Synthesis and characterization

The preparation of TiO₂-modified PPy molecular imprinting polymer began with the surface modification of TiO₂ nanoparticles. A suspension of TiO₂ nanoparticles (1.0 g) in ethanol (50 mL) was subjected to ultrasonication for 30 min to achieve uniform dispersion. The suspension was then mixed with 3-aminopropyltriethoxysilane (2.0 mL) and refluxed at 80°C for 4 h under nitrogen atmosphere. The synthesized TiO₂ nanoparticles were isolated through centrifugation, subsequently rinsed multiple times with ethanol, and then dehydrated in a vacuum oven at 60°C for a 12-h period.

The synthesis procedure entailed establishing a preliminary interaction between CIN and pyrrole within a phosphate-based aqueous medium, supplemented with functionalized TiO₂ nanoscale particles under specific pH and concentration conditions. The solution was agitated for half an hour at ambient conditions to promote intermolecular hydrogen bond establishment. Utilizing a standard electrochemical cell with three electrodes – glassy carbon as the working electrode, platinum wire serving as the counter electrode, and silver/silver chloride as the reference electrode – the polymer synthesis was performed. The electropolymerization process involved cyclic voltammetric scanning from −0.2 to +0.8 V, applying a 50 mV·s⁻¹ sweep rate across 10 consecutive cycles.

2.2 Electrode fabrication

Gold electrodes were fabricated via semi-automatic screen printing techniques employing a specialized template design. The modification of screen-printed electrodes began with electrochemical cleaning by cycling in 0.5 M H₂SO₄ between −0.2 and +1.3 V at 100 mV·s⁻¹ until stable voltammograms were obtained. The MIP solution was drop-cast (5 µL) onto the working electrode surface and allowed to dry at room temperature. Template removal was achieved through potential cycling in 0.5 M oxalic acid solution between 0.0 and +1.0 V at 100 mV·s⁻¹ for 15 cycles.

3.1 Material characterization

The surface morphology and structural features of TiO₂ nanoparticles, functionalized TiO₂, and the MIP composite were examined in detail. Scanning electron microscopy (SEM) analysis revealed the distinct morphological evolution during the sensor fabrication process. Figure 1a shows pristine TiO₂ nanoparticles exhibiting a uniform spherical morphology with an average particle diameter of 25 nm. Following surface functionalization, the TiO₂ nanoparticles maintained their spherical structure while displaying slight aggregation tendencies (Figure 1b).

Figure 1

Electron microscopy analysis of sensor materials: (a) SEM image of pristine TiO₂ nanoparticles and (b) SEM image of functionalized TiO₂ nanoparticles.

X-ray diffraction (XRD) patterns were recorded to investigate the crystalline structure of the materials. Figure 2 presents the XRD patterns of the synthesized composites. The characteristic peaks at 2θ values of 25.3, 37.8, 48.0, 53.9, and 55.1° correspond to the (101), (004), (200), (105), and (211) crystal planes of anatase TiO₂, respectively. The preservation of these peaks in the MIP composite confirms the stability of the TiO₂ crystal structure during the modification process [26]. A broad peak observed between 20° and 30° in the composite pattern indicates the presence of the amorphous PPy matrix [27].

Figure 2

XRD patterns of pristine TiO₂ nanoparticles and molecularly imprinted polymer composite. Major crystallographic planes are indexed.

Fourier transform infrared (FTIR) spectroscopy provided valuable information about the chemical composition and interactions within the composite material. Figure 3 displays the FTIR spectra of the various synthesis stages. The characteristic Ti–O–Ti stretching vibration at 495 cm⁻¹ and Ti–O stretching at 625 cm⁻¹ confirm the presence of TiO₂ [28]. After surface functionalization, new bands appeared at 1,085 and 1,560 cm⁻¹, corresponding to Si–O–Si and N–H bending vibrations, respectively. The molecular imprinted composite exhibited additional peaks at 1,455 cm⁻¹ (C–N stretching) and 1,640 cm⁻¹ (C═C stretching), characteristic of the PPy structure [29].

Figure 3

FTIR spectra of TiO₂ nanoparticles and molecularly imprinted polymer composite showing characteristic vibrational bands.

Thermal stability analysis was conducted using TGA under nitrogen atmosphere. Figure 4 illustrates the thermal decomposition profiles of the materials. The TiO₂ nanoparticles showed minimal weight loss (<8%) up to 800°C, confirming their high thermal stability. The functionalized TiO₂ exhibited a weight loss of approximately 8% between 200°C and 400°C, attributed to the decomposition of surface modifiers. The molecularly imprinted composite demonstrated a three-stage decomposition pattern: initial moisture loss (30–150°C), template removal (150–300°C), and polymer degradation (300–600°C), with a total weight loss of 35%.

Figure 4

TGA curves of TiO₂ nanoparticles and molecularly imprinted polymer composite under nitrogen atmosphere (heating rate: 10°C·min⁻¹).

3.2 Electrochemical characterization

The electrochemical behavior of the developed sensor was systematically investigated using various electrochemical techniques to understand the electron transfer characteristics and surface properties of the modified electrode. Cyclic voltammetry studies using [Fe(CN)₆]^3−/4− as a redox probe revealed the progressive modification of the electrode surface. Figure 5a presents the cyclic voltammograms obtained at different stages of sensor fabrication. The bare screen-printed gold electrode exhibited well-defined redox peaks with a peak-to-peak separation (ΔE _p) of 147 mV, indicating reversible electron transfer kinetics [30]. Following TiO₂ modification, the peak currents increased by approximately 45%, attributed to the enhanced electroactive surface area provided by the nanoparticles. The molecular imprinting process initially resulted in decreased peak currents and increased ΔE _p (422 mV) due to the insulating nature of the polymer layer. However, after template removal, the peak currents partially recovered, and ΔE _p decreased to 327 mV, suggesting the formation of accessible binding cavities [31].

Figure 5

Electrochemical characterization of sensor fabrication steps: (a) cyclic voltammograms in 5 mM [Fe(CN)₆]^3−/4− containing 0.1 M KCl at 50 mV·s⁻¹ for bare electrode, TiO₂-modified electrode, molecular imprinted polymer before template removal, and after template removal, and (b) Nyquist plots with corresponding for the same modification steps.

Electrochemical impedance spectroscopy provided valuable insights into the interfacial properties of the modified electrodes. The Nyquist plots (Figure 5b) were fitted using an equivalent circuit model (inset of Figure 5b). The bare electrode displayed a small semicircle, indicating efficient electron transfer. The TiO₂ modification decreased Rct, consistent with improved conductivity. The MIP layer significantly increased [32], while template removal reduced, corroborating the CV findings.

Cyclic voltammetry was employed to determine the electrochemically active surface area by applying the Randles–Sevcik relationship across different potential sweep velocities. The graph depicts a direct proportional correlation between maximum electrical currents and the square root of scanning velocity across various electrode surface treatments. The calculated electroactive surface areas were 0.052, 0.075, and 0.068 cm² for bare, TiO₂-modified, and molecularly imprinted electrodes, respectively. These results indicate a 44% increase in surface area after TiO₂ modification, with a slight decrease following molecular imprinting due to polymer coverage [33] (Figure 6).

Figure 6

Plot of peak current versus square root of scan rate for different electrode modifications.

Electron transfer kinetics were evaluated by analyzing the relationship between ΔE _p and scan rate. Employing the Nicholson approach, researchers determined the electron transfer kinetics across different interfaces by evaluating the rate constant for heterogeneous electron transfer. The bare electrode exhibited ks = 5.8 × 10⁻³ cm·s⁻¹, which increased to 8.2 × 10⁻³cm·s⁻¹ after TiO₂ modification. The molecularly imprinted sensor showed ks = 3.4 × 10⁻³ cm·s⁻¹, reflecting the balance between polymer insulation and facilitated electron transfer through binding cavities [34].

To elucidate the electrochemical oxidation mechanism of CIN, we considered its structural features, particularly the presence of a tertiary aromatic amine group, which is typically responsible for redox activity. Upon applying a positive potential, CIN undergoes a one-electron oxidation process at the nitrogen atom, leading to the formation of a radical cation intermediate. To confirm the number of electrons transferred (n), we employed the Laviron method by analyzing the linear relationship between peak potential (E _p) and the logarithm of scan rate. The slope of this plot was approximately 59.2 mV/decade, consistent with a single-electron transfer process (n ≈ 1) under the assumed transfer coefficient (α = 0.5). This is in agreement with the literature on structurally similar amine-containing compounds [25]. The oxidation reaction can be summarized as

These findings corroborate the voltammetric data presented, where well-defined oxidation peaks were observed and used for analytical quantification. The selective recognition achieved through molecular imprinting further confirms that the redox activity is specific to CIN, likely involving its electroactive aromatic amine moiety.

3.3 Optimization studies

The analytical performance of the molecularly imprinted sensor was systematically optimized by investigating various experimental parameters that influence the detection of CIN. The effect of pH on the electrochemical response was studied over the range of pH 3.0–9.0 using phosphate buffer solutions. Figure 7a illustrates the relationship between peak current and pH for 50 μM CIN. The current response increased significantly from pH 3.0 to 5.0, reached maximum intensity at pH 5.0, and gradually decreased at higher pH values. The peak potential shifted linearly with pH (slope = −59.2 mV/pH), suggesting a proton-coupled electron transfer process [35,36]. The optimal response at pH 5.0 can be attributed to the balance between the protonation state of CIN and the stability of the polymer matrix [37].

Figure 7

Optimization of experimental parameters: (a) effect of pH on peak current response for 50 μM CIN. Inset shows the linear relationship between peak potential and pH. (b) Linear relationship between peak current and the square root of scan rate.

Scan rate studies provided insights into the mass transport characteristics of the sensor. The peak current for oxidation demonstrated a linear correlation with the scan rate’s square root, suggesting that the mechanism is governed by diffusive transport (Figure 7b). The peak potential shifted positively with increasing scan rate, with a slope of 28.3 mV/decade, suggesting quasi-reversible electron transfer kinetics [38].

The binding kinetics of CIN to the molecularly imprinted sites were investigated through time-dependent current response measurements [39,40]. Figure 8a shows the evolution of peak current with incubation time at different CIN concentrations. The binding process followed pseudo-first-order kinetics, with equilibrium reached within 15 min for concentrations below 100 μM [41]. The association rate constant (ka) was determined to be 3.2 × 10⁴ M·s⁻¹, while the dissociation rate constant (kd) was 1.8 × 10⁻³s⁻¹, yielding an affinity constant (Ka) of 1.78 × 10⁷ M⁻¹.

Figure 8

Kinetic and thermodynamic studies: (a) time-dependent current response at different CIN concentrations (10–100 μM) showing binding kinetics and (b) temperature dependence of peak current. Inset shows the Arrhenius plot for electron transfer activation energy determination.

Temperature effects on sensor performance were evaluated between 15°C and 45°C. Figure 8b demonstrates the variation in peak current and potential with temperature. The current response increased by approximately 2.5%/°C up to 35°C, followed by a decline at higher temperatures [42]. This behavior reflects the competition between enhanced diffusion kinetics and decreased stability of the molecular imprinting sites at elevated temperatures [43]. The activation energy for the electron transfer process was calculated to be 28.3 kJ·mol⁻¹ using the Arrhenius plot (inset).

These optimization studies established optimal operating conditions of pH 5.0, scan rate 50 mV·s⁻¹, incubation time 15 min, and temperature 25°C for subsequent analytical applications of the sensor.

3.4 Analytical performance

The practical utility of the molecularly imprinted sensor was evaluated through comprehensive analytical performance studies under optimized conditions. Differential pulse voltammetry was employed to establish the quantitative relationship between current response and CIN concentration. Figure 9a presents overlaid voltammograms showing well-defined oxidation peaks at increasing CIN concentrations. The peak current exhibited two distinct linear ranges (Figure 9b): 0.1–10 and 10–100 μM, with regression equations of ip (μA) = 0.542C (μM) + 0.128 (R ² = 0.9985) and ip (μA) = 0.326C (μM) + 2.314 (R ² = 0.9962), respectively. The dual-linear ranges likely reflect the heterogeneous binding site distribution within the MIP, with high-affinity sites dominating at lower concentrations. The limit of detection (LOD) was determined to be 0.035 μM (S/N = 3), while the limit of quantification was 0.12 μM (S/N = 10). These values demonstrate superior sensitivity compared to conventional electrochemical methods and are suitable for clinical applications.

Figure 9

Analytical performance characterization: (a) differential pulse voltammograms at increasing CIN concentrations (0.1–100 μM) and (b) calibration curves showing dual-linear ranges with corresponding regression equations.

Reproducibility studies encompassed both intra-sensor and inter-sensor precision. Figure 10a shows the current responses obtained from six consecutive measurements using a single sensor, yielding an relative standard deviation (RSD) of 2.8% for 50 μM CIN. Inter-sensor reproducibility was evaluated using six independently fabricated sensors, producing an RSD of 3.9%. These results confirm the reliability of the sensor fabrication process and measurement protocol. Long-term stability was assessed by monitoring sensor response over 30 days, with electrodes stored at 4°C between measurements. Figure 10b illustrates the retention of sensor activity, maintaining 95.2% of the initial response after 30 days. The excellent stability can be attributed to the robust molecular imprinting architecture and the stabilizing effect of TiO₂ nanoparticles.

Figure 10

Precision and stability studies: (a) reproducibility data showing intra-sensor (n = 6) and inter-sensor (n = 6) measurements for 50 μM CIN and (b) long-term stability assessment over 30 days with electrodes stored at 4°C.

Interference studies evaluated the sensor’s selectivity against potential competing species commonly found in clinical samples. Table 1 presents the tolerance ratios for various interferents, determined as the maximum concentration ratio ([interferent]/[CIN]) causing less than ±5% change in signal. The sensor exhibited excellent selectivity, tolerating 100-fold excess of common ions (Na⁺, K⁺, Cl⁻, HCO₃ ⁻) and 50-fold excess of structurally similar compounds (procaine, lidocaine, benzocaine).

3.5 Real sample analysis

The practical applicability of the molecularly imprinted sensor was rigorously evaluated through analysis of serum samples and comparison with established analytical methods. Initial studies focused on method optimization to minimize matrix effects. A simple 1:1 dilution with phosphate buffer (pH 5.0) followed by centrifugation proved sufficient for reliable measurements, eliminating the need for complex extraction procedures. We evaluated the accuracy of the analytical method by introducing predetermined amounts of CIN into serum specimens and assessing the subsequent detection rates. Table 2 summarizes the recovery results obtained from twenty different serum samples spiked at three concentration levels (1.0, 10.0, and 50.0 μM). The average recoveries ranged from 97.2% to 102.3%, with relative standard deviations below 4.5%.

Precision studies encompassed both intra-day and inter-day variations. Table 3 presents the results obtained from analyzing quality control samples at three concentration levels over five consecutive days. The intra-day relative standard deviations ranged from 2.1% to 3.8%, while inter-day values were between 3.2% and 4.7%, demonstrating excellent method precision.

3.6 Comparative analysis with previous methods

To further contextualize the analytical performance of the proposed sensor, we compiled a comparative table (Table 4) that benchmarks our method against several previously reported electrochemical and potentiometric strategies for CIN detection. The comparison includes detection limits, linear ranges, matrix compatibility, and sample preparation requirements. As shown in Table 4, many conventional approaches – such as potentiometric ion-selective electrodes or CNT-based sensors – offer reasonable sensitivity but often require complex fabrication, extensive calibration steps, or use of costly modifiers. Notably, some rely on PVC membranes or ionic liquids that are not ideal for point-of-care applications due to leaching or biocompatibility concerns. In contrast, our MIP-based electrochemical sensor not only demonstrates a competitive detection limit (0.035 μM) and dual-linear ranges but also features a rapid, low-cost fabrication process, simplified sample preparation (1:1 dilution and centrifugation), and high selectivity in biological matrices. These attributes support its practical applicability for clinical point-of-care use.