Characterization of dual-MIP-PmDB/PoAP-GCE
The affirmation of the configuration of the dual-MIP film on GCE was investigated through the SEM technique (Fig. 2). The dual-MIP-PmDB/PoAP-GCE presented a rough surface and was covered via many slices (Fig. 2A), illustrating the successful modification of MIP film. The rough surface provided a wide particular surface area for the detection and recognition of AA and Tyr. As exhibited in Fig. 2B, after eluting the dual template, the MIP surface is rough and full of many cavities, but the slices disappeared, indicating that the templates were extracted from the polymer layer. A comparison of the dual-MIP film morphology before and after the elimination of the dual analyte highlights the architecture of copious imprinted sites following the elimination step.
Additionally, before and after extracting the dual template, FTIR spectra was performed to further evaluation the accuracy of the sensor (Fig. 3). As anticipated, the characteristic bonds of the dual-MIP layer were declared in all substances. The bond remarked at 3446 cm− 1 related to the OH, its intensity decreased after dual analyte elimination, providing the interaction of monomers and analytes. The bond shown at 2458 cm− 1 is proportion to the carbonyl region, which its intensity was decreased after the removal of analytes compared to before. The peak at 1735 cm− 1 is related to the Ester group. It has been decreased after the washing step. The bond that appears at 1483 cm− 1 for C = C belongs to the aromatic ring bending vibrations, decreasing peaks intensity in leached polymer severely that proved dual template elimination. The achieved results of the FTIR analysis assured that there were not both AA and Tyr in the leached dual-MIP film.
Electrochemical characterization of the modified electrodes
The CV behaviour of the various modified electrodes was recorded in a 5 mM [Fe(CN)6]3−/4− probe (Fig. 4A). After the electropolymerization stage, the peak current of dual-MIP has been greatly reduced as compared to the bare GCE (curve a), illustrating that the electropolymerization has been favorably coated on the electrode (curve b) and this has caused the blocking of the electron transfer from the solution. After eliminating the dual template via the eluting solution, because sites were created on the MIP layer to transfer the electrons, the current response reappeared (curve c) as compared to curve b. Subsequently, the NIP-PmDB/PoAP-GCE was evaluated (curve d), indicating the insulating characterization of the NIP layer and an extreme reduction in the current response.
EIS method was applied to characterize the modified electrodes using the probe of 5 mM [Fe(CN)6]3−/4− (Fig. 4B). The charge transfer resistance (Rct) values severely incremented from 305.2 Ω (curve a) to 13164.7 Ω (curve b) when the dual-MIP film was deposited on GCE because the dual-MIP layer led to the lower transfer of the electron. The Rct was reduced to 3823.5 Ω after templates extraction (curve c) due to the making of particular recognition cavities that decreased the resistance to the transfer of electrons. Lastly, the resistance incremented (Rct = 7385.4 Ω) for NIP-PoAP/PmDB-GCE (curve d) because the polymeric matrix conductivity was reduced. The obtained results of EIS were in accordance with CV measurements.
Optimization of the factors for MIP sensor preparation
To obtain an incomparable output of the designed dual-MIP sensor; a series of factors, including cycle numbers, template/monomer ratio, and pH value of PBS were evaluated.
The scanning cycles, an important parameter, can sorely affect the thickness of the dual-MIP film. Figure 5A illustrates the DPV current responses of 10 µM AA and 1 µM Tyr (curve a and curve b) that were achieved from a dual-MIP sensor in various electropolymerization scanning cycles. It is exhibited when the cycle number was 10, the maximum current responses of analytes were obtained. Besides, the peak currents of dual template on the MIP layer firstly increased and then decreased, when the cycle number was over 10 the thicker MIP layer was achieved, and when it was less than 10, the thinner MIP film resulted. As the result, 10 cycle was selected as the optimal number of cycles.
To optimize the mole ratio of dual template to monomers, because it has a significant role for the MIP structure and the rebinding affinity, various template/monomer ratios from 1:10 to 1:30 were appraised while the concentration of the analytes was kept at 0.05 mM, and the DPV current responses of the dual-MIP-PmDB/PoAP-GCE after every electropolymerization was taken. According to Fig. 5B, the current response increased with the template/monomer ratio, until it remained constant at a 1:20 ratio. Thus, the template/monomer ratio was chosen as 1:20 for dual-MIP synthesis.
The pH as another effective parameter was investigated since its value affects the detection ability, the interaction between the monomers and dual template, and the peak current response. Figure 5C affirmed the DPV current responses of 10 µM AA and 1 µM Tyr (curve a and curve b) achieved at various pH values from PBS. Here, as the pH incremented to 6-7.4, the peak current of templates on the dual-MIP layer increased, and, then, decreased with an extra increasing the pH value. Then, pH = 7.4 of PBS was used for the electrochemical recognition.
Electrochemical properties of AA and Tyr on the modified electrodes
The CV curves of AA and Tyr on the modified electrodes in PBS (pH = 7.4) shows in Fig. 6. At the bare electrode, petty oxidation peaks were illustrated for AA and Tyr due to the slow rate of electron transfer (curve a). After electropolymerization, at dual-MIP-PmDB/PoAP modified electrode (curve b), the current responses of analytes increased severely which can be attributed to the synergistic electrocatalytic issue on the dual-MIP film. The dual-MIP layer provided the particular binding holes that improve the absorption efficiency of analytes on GCE. In other words, the dual template can be easily absorbed in the proper cavities in the MIP film, the electron transfer between the dual target and electrode was also accelerated and resulted in the enlarged current responses. A comparison of the NIP electrode was also assayed (curve c). After modification with NIP, weak oxidation peaks were resulted due to the lack of particular binding holes for the analytes.
Selective detection from AA and Tyr
In order to confirm the feasibility of the simultaneous detection of AA and Tyr through the dual-MIP-PmDB/PoAP-GCE, the DPVs performed in the mixtures when the concentration of one analytes changed and another was held constant. Figure 7A indicates that the peak current for the AA increases linearly with the increment its concentration, while the peak response for Tyr hold almost unchanged (Tyr: 60 µM). This exhibits which Tyr has no interfering effect on the determination of AA. Similarly, Fig. 7B displays that with the increase in Tyr concentration, the peak responses of Tyr shows a linear increase, while the peak responses for AA are nearly constant (AA: 100 µM), which proposes that the presence of AA do not interfere with the response of Tyr. All the results severely signify that AA and Tyr can be individually determined in their mixture using the suggested technique.
Analytical application
The analytical application of the dual-MIP sensor was examined via the simultaneous detection of AA and Tyr. Figure 8A shows DPV responses of the dual analyte on the dual-MIP sensor, illustrating that the oxidation current responses of AA and Tyr incremented simultaneously with the enhancing concentrations. As result, the peak currents were proportional to AA and Tyr concentrations in the ranges of 0.1–300 µM and 0.01–180 µM, respectively. The linear regression equations were described as I (µA) = 0.567 (µM) + 5.1989 (R2 = 0.9989) for AA, and I (µA) = 0.921 (µM) + 10.5072 (R2 = 0.9992) for Tyr (Fig. 8B and Fig. 8C). In this study, the detection limit (LOD) was obtained based on S/N = 3. The LOD was calculated as 0.03 µM for AA and 0.003 µM for Tyr. Then, the dual-MIP sensor displayed two linear regression equations at different concentration ranges with low LODs. Moreover, Comparative results of the dual-MIP sensor and formerly reported studies are summarized in Table 1. The introduced sensor displayed a wider linear range and lower LODs for the simultaneous determination of AA and Tyr
Selectivity, repeatability, reproducibility, and stability of the sensor
The selectivity was investigated via the DPV technique of 10 µM of AA and Tyr in the presence of 100-fold of various interference compounds with the same properties or structures such as sucrose, glucose, fructose, urea, citric acid, and starch. As exhibited in Fig. 9, the high peak currents to AA and Tyr at dual-MIP-PmDB/PoAP-GCE without apparent responses to the interfering compounds indicates that the dual template can be recognized in presence of interference substances because the imprinted membranes were the specific detection cavities to the analytes and the other compounds have no effect on the detection of AA and Tyr, indicating that the dual-MIP sensor possesses a good anti-interference characteristic in the determination of AA and Tyr so the admissible selectivity of this sensor was evolved from the MIP film that can be related to the particular binding cavities on it.
Repeatability was measured via detecting 10 µM of AA and Tyr for 10 measurements on the same designed sensor. Gratifying repeatability was achieved with the relative standard deviation (RSD, n = 10) of 3.4% and 2.9% for AA and Tyr, respectively. The reproducibility was obtained through detecting 10 µM of AA and Tyr using 5 various MIP sensors prepared under the same experimental conditions. The RSD was calculated to be 2.6% and 3.5% for AA and Tyr, respectively, showing good reproducibility of the sensor. In addition, more than 90% of the initially sensor response was retained after placing for 21 days at 5°C, indicating the suitable stability of the sensor.
Sample analysis
The dual-MIP-PmDB/PoAP sensor was applied in the recognition of AA and Tyr in serum samples by the DPV technique and standard addition method to illustrate the applicability of the present technique. The samples were treated as Section 2.3 and any sample undergoes three measurements. The results were briefed in Table 2. The recoveries were from 97 to 105% and the RSD was less than 3.5% which exhibited the usability of this sensor in the actual sample.