Results and discussion

Characterization of complex

Figure 2 displays the FT-IR spectra of [Cu(opd)2(H2O)(μ-SCN)Ni(opd)(SCN)3]. The amine (NH2) group in the free ligand o-phenylenediamine was found to stretch at a frequency of around 1449 cm-1 [17]. This spectrum was detected in the combination with [Cu(opd)2(H2O)(μ-SCN)Ni(opd)(SCN)3] at around 1339 cm-1. This finding suggests that the stretching frequency of the NH2 functional group has decreased from that of the free ligand to that of the complex, which may be due to coordination of the NH2 group to copper and nickel ions. Because nitrogen and metal share the non-bonding pair of electrons of the NH2 group during the coordination process, the electron density on nitrogen lowers, which in turn decreases the electron density in the N–H bond.

Figure 2.: FT-IR spectrum of [Cu(opd)2(H2O)(μ-SCN)Ni(opd)(SCN)3)]

The C=C stretching frequency of the aromatic ring in free o-phenylenediamine was observed between 1640 and 1680 cm-1, matching the complex's absorption at approximately 1660 cm-1. The stretching frequency of the aromatic C-H groups in free ligand o-phenylenediamine was about o-phenylenediamine in the complex [Cu(opd)2(H2O)(μ-SCN)Ni(opd)(SCN)3]. The 1050 absorption band is due to C-N bond stretching vibrations because nitrogen is bonded to the metal through its lone pair of electrons. A strong absorption peak at 1760 cm-1 exists as the C=S bond of the thiocyanate ligand stretches while the thiocyanate ligand connects to nickel metal through its nitrogen atom as a terminal ligand [18].

UV-Vis spectra of the complex [Cu(opd)2(H2O)(μ-SCN)Ni(opd)(SCN)3], which appeared in Figure 3, were recorded at two different concentrations because of the existence of two electronic transitions: ultraviolet transitions at 10-5 M and visible transitions at 10-3 M, both in dimethylformamide solvent [19]. The electronic spectrum of this complex shows two absorption bands at 380 nm and 445 nm due to d-d transitions of the nickel(II) ion [19].

Fig. 3.: UV-Vis spectra of [Cu(opd)2(H2O)(μ-SCN)Ni(opd)(SCN)3)]

The absorption bands at 210, 225 and 290 nm are related to intraligand transitions n→π*, π→π*, which occur in the orthophenylene diamine ligand and thiocyanate ligand. The electronic spectrum of this complex shows a single absorption band at 425 nm, which corresponds to d-d transitions of copper(II) ion [20].

Synthesis of nano-complex

25 milliliters of DMF solution were used to dissolve 0.50 mM of [Cu(opd)2(H2O)(μ-SCN)Ni(opd)(SCN)3]. After that, it was ultrasonicated for around half an hour. Following that, the compound's solvothermal reaction was conducted in an oven-mounted autoclave at 120 °C for 48 hours. The precipitates were collected after the solvothermal reaction was complete and the autoclave had cooled to room temperature. Lastly, the [Cu(opd)2 (H2O)(μ-SCN)Ni(opd)(SCN)3] were made by repeatedly washing the precipitates with DMF and ethanol and drying them overnight at 100 °C. After filtration, the precipitate was allowed to dry open. mp: 245 °C; yield: 87 %.

Characterization of nano complex of [Cu(opd)2(H2O)(μ-SCN)Ni(opd)(SCN)3)]

Figure 4 shows the XRD pattern of the nano-sized compound, which was acquired using single-crystal X-ray diffraction. The complex's composition and crystalline phase are revealed by the XRD pattern. The diffraction peak widths indicate that the particles in the nanocrystal complex are nanoscale. The Debye-Scherrer equation of the nanocrystal complex (a) yielded an average diameter of around 56 nm.

Figure 4.: XRD pattern of the nano-complex [Cu(opd)2(H2O)(μ-SCN)Ni(opd)(SCN)3)]

Figure 5 displays the FE-SEM image of the [Cu(opd)2(H2O)(μ-SCN)Ni(opd)(SCN)3]. The SEM image show that the nano-complex crystals are produced in spheres with smooth surfaces.

Figure 5.: FE-SEM images of the nano-complex [Cu(opd)2(H2O)(μ-SCN)Ni(opd)(SCN)3)]

Electrochemical response of Cu-Ni/SPGE compared to unmodified SPGE for IRN determination

Cyclic voltammograms of 1.0 μM IRN recorded at the unmodified SPGE and the Cu-Ni/SPGE in phosphate buffer solutions are shown. The cyclic voltammogram, which showed an oxidation peak at 790 mV with an _I_pa of 2.7 μA, indicated moderate reactivity of the unmodified SPGE toward IRN oxidation. Nonetheless, a clear IRN oxidation peak was visible in the Cu-Ni/SPGE. Apart from the notable rise in IRN's _I_p = 6.9 μA, the SPGE's surface was modified to lower the anodic peak potential (_E_p = 690 mV).

Figure 6.: The CV responses of 1.0 μM IRN/PBS at unmodified SPGE and Cu-Ni/SPGE are shown. Scan rate 50 mV s-1

Impact of scan rate

The Cu-Ni/SPGE response to the oxidation of 1.0 μM IRN in buffer solution was recorded at various scan rates (Figure 7). The CVs shown in Figure 7 indicate that the current height increases gradually with increasing scan rate. The proper linearity between _I_pa and _v_1/2 in Figure 7 (Inset) indicates diffusion-controlled electrooxidation of IRN on Cu-Ni/SPGE.

Figure 7.: The CV responses of Cu-Ni/SPGE at different scan rates in the buffer solution containing 1.0 μM IRN. Inset: Ip and v1/2 have a linear relationship

Chronoamperometric investigations of IRN at Cu-Ni/SPGE

The oxidation of IRN on the Cu-Ni/SPGE was further investigated using chronoamperometry. The chronoamperograms shown in Figure 8 were recorded on the Cu-Ni/SPGE for different IRN concentrations in the buffer solution using a step potential to 740 mV. The acquired chronoamperograms showed that increases in anodic current were associated with higher IRN concentrations. In chronoamperometric studies, the diffusion coefficient (D) of electroactive compounds can be computed using the Cottrell Equation (1):

Figure 8.: chromatograms for the oxidation of IRN concentrations on the Cu-Ni/SPGE

The Cottrell plots (I-t−1/2 curves) displayed a linear relationship over a certain time period (0.1 to 3.0 s) (Figure 9A). The Cottrell curves were generated from chronoamperograms recorded at a specific IRN concentration. Next, the slope of the Cottrell curves was plotted against the different concentrations of IRN (Figure 9B). Finally, using Cottrell's equation and the slope of the resulting figure in Figure 9B, the D for IRN was found to be 5.4×10−5 cm2 s-1.

Figure 9.: (A) Chronoamperogram plots of I vs. t−1/2; (B) a plot of the straight-line slope against IRN concentration

Electroanalysis performance of Cu-Ni/SPGE for IRN

The differential pulse voltammetry (DPV) was used to quantitatively assess IRN on the Cu-Ni/SPGE. The DPVs of Cu-Ni/SPGE at different IRN concentrations in the buffer solution are displayed in Figure 10. As the IRN concentration increased, a linear relationship between the _I_pa and IRN concentration was seen (linear range: 0.01 to 3.0 μM) (Figure 11). Furthermore, the calculated LOD of the Cu-Ni/SPGE for IRN was 0.003 μM.

Figure 10.: Cu-Ni/SPGE responses to different IRN concentrations in the buffer solution

Figure 11.: Calibration plot of IRN at Cu-Ni/SPGE

Stability, reproducibility, and repeatability studies of Cu-Ni/SPGE

The Cu-Ni/SPGE testing process evaluated its stability over a 15-day storage period at room temperature. The sensor measured its current response to 1.0 μM IRN in a buffer solution every 3 days, reaching the 15-day testing period. The peak current after 15 days decreased by 4.1 % from the sensor's original measurement. The DPV technique was used to assess repeatability by measuring the response current of five modified SPGEs in a buffer solution containing 1.0 μM IRN. The electrodes produced current responses, resulting in a relative standard deviation (RSD) of less than 3.3 %. Ten consecutive voltammetric (DPV) tests with 1.0 μM IRN were performed to evaluate the repeatability of the Cu-Ni/SPGE response. After ten measurements, 96.1 % of the initial response current was retained. The results proved that the Cu-Ni/SPGE sensor demonstrated excellent repeatability and maintained its performance across different testing conditions.

Real sample analysis

Standard addition methods were used to assess IRN levels in injection samples and evaluate the applicability of the Cu-Ni/SPGE sensor to real samples. The results are presented in Table 1. Recovery of IRN increases from 97.1 to 104.5 %. The study found that IRN detection in samples could be achieved through the developed sensing platform.

Spiked concentration, μM | Found Concentration, μM | Recovery, % | RSD, %
0 | 0.19 | - | 3.1
0.5 | 0.67 | 97.1 | 2.4
0.7 | 0.93 | 104.5 | 1.5
0.9 | 1.08 | 99.1 | 2.3
1.1 | 1.31 | 101.5 | 2.7

In-vitro antimicrobial experiment

The antibacterial activity of the complex was tested against two Gram-positive bacteria, Staphylococcus aureus (S. aureus ATCC 25923) and Enterococcus faecalis (Enter_faeca ATCC 29212), and two Gram-negative bacteria, Escherichia coli (E. coli ATCC 25922) and Pseudomonas aeruginosa (P. aeruginosa ATCC 27853). The broth macro-dilution method was used to determine both minimum bactericidal concentration and minimum inhibitory concentration of the nano-complex (a) (Table 2). The measurement process included determining the size of every bacterial-growth-free inhibition zone (IZ) (Table 2). Three separate MIC, MBC, and inhibitory zone tests were performed before averaging the results. The testing process used amikacin and gentamycin as standard antibacterial agents, which had been tested to establish their effectiveness [21].

 | opd | Nano-complex
MIC, mg mL-1 | MBC, mg mL-1 | MIC, mg mL-1 | MBC, mg mL-1
Escherichia coli | 0.0122 | 0.0247 | 0.0121 | 0.0248
Pseudomonas aeruginosa | 0.0244 | 0.0498 | 0.0123 | 0.0244
Staphylococcus aureus | 0.0122 | 0.0249 | 0.0060 | 0.0122
Enterococcus faecalis | 0.0122 | 0.0248 | 0.0028 | 0.0057

 | Inhibition zone, mm
opd | Nano-complex | Gentamycin | Amikacin
Escherichia coli | 17 | 22 | 19.2 | -
Pseudomonas aeruginosa | unavailable for the study | 22 | 20.1 | -
Staphylococcus aureus | 17 | 28 | - | 18.8
Enterococcus faecalis | unavailable for the study | 26 | - | 19.2