Effect of palladium and its nanogeometry on the redox electrochemistry of tetracyanoquinodimethane modified electrode; application in electrochemical sensing of ascorbic acid

The current work reports the effect of palladium nanoparticles and their nanogeometry on the redox electrochemistry of tetracyanoquinodimethane (TCNQ) modified electrodes. Palladium nanoparticles were prepared with different concentrations of 3-aminopropyltrimethoxysilane and calcination at 600 °C to yield PdNP-1 and PdNP-2 of the average size of 1 µm and 12 nm, respectively. The palladium nanoparticles were characterized by TEM, XRD, and AFM techniques. The electrochemical excellence of ascorbic acid was resoluted using cyclic voltammetry amperometry, electrochemical impedance spectroscopy, and differential pulse voltammetry. A limit of detection (LOD) was found to be 51.61, 44.38 and 30.10 µM over a linear range from 50 to 625 µM for modified CPE/TCNQ, CPE/TCNQ-PdNP-1, and CPE/TCNQ-PdNP-2, respectively, determined by amperometric analysis for ascorbic acid at pH 7. The synergistic effect of Palladium and π* orbital of TCNQ played an important role in the enhancement of the catalytic activity of the modified electrode. The modified electrode showed good sensitivity, stability, and reproducibility which was confirmed by cyclic voltammetry, and amperometric analysis. The charge transfer resistance value also indicated that the modified electrode hads good electrocatalytic activity.


Introduction
Nanotechnology has brought about a standard shift in the essential characteristics of matter during the last few decades, leading to the creation of novel systems and materials. Nanodevices are highly sought-after on the market because of their sensitivity, low power consumption, selectivity, precision, efficiency, and fast speed [1]. As significant, though, is the development of an electrochemical sensing platform that is very sensitive, excellently selective in the attendance of other biomolecules, has a long shelf life, is stable, and exhibits minimal cytotoxicity without the use of an electron transfer mediator [2][3][4]. Numerous chemically modified electrodes and biosensors have been developed and successfully used in the detection of numerous biomolecules and toxic metals using a variety of electrochemical sensing techniques, including chronoamperometry, electrochemical impedance spectroscopy, cyclic voltammetry, linear sweep voltammetry, and square wave voltammetry [5,6]. Carbon paste working electrodes have recently been incorporated into electrochemical sensors for the determination of significant electroactive molecules due to their distinctive benefits, such as enhanced electronic properties, low cost, renewability of the electrode surface, the flexibility of chemical modification, wide potential windows, and low background current [7,8]. The use of nanomaterials in numerous sectors is expanding quickly and has promising futures. The development of sensitive and selective biosensors has benefited from recent advancements in nanotechnology [9]. The electrochemical reactivity and sensitivity of analytes can be increased by altering the electrode surface using a variety of nanostructures [10]. Redox-active sites increase the speed of electron transport by lowering its activation overpotential [11,12]. It should be noted that because of their large surface areas, excellent electrical conductivity, and strong catalytic activity, noble metal NPs have been explored for electrochemical sensing. These metal NPs are helpful to lower the overpotentials of electrochemical sensors and keep redox reactions reversible [13,14].
Electroanalytical techniques are valuable for gaining a thorough knowledge of multi-electron transfer systems. The electrochemistry of neurotransmitters, important biomolecules, organic (insecticides and pesticides), and inorganic materials, among others, is rife with multi-step mechanisms [10]. These techniques have a wide linear dynamic range, great sensitivity, precision, and accuracy, and need apparatus that is comparatively inexpensive. Electroanalytical investigations are more frequently utilized in industrial, and biomedical applications, drug analysis in various dosage forms, and notably in biological samples with the development of more sensitive pulse techniques [15]. Electroanalytical techniques may quickly address numerous pharmaceutical issues with great, precision, sensitivity, accuracy, and selectivity. Some of the most efficient electroanalytical methods are based on the principle of continually adjusting the applied potentials to the electrode solution contact and the consequently studied current. Electroanalytical techniques (particularly stripping analysis) are well-established as effective methods for determining trace analytes. These methods have been developed for a wide range of cations, anions, as well as organic compounds. Electrochemical methods, particularly voltammetry, have grown in prominence in recent years. These electrochemical methods have been used to identify pharmaceutical chemicals in dosage forms as well as biological samples [9].
Tetracyanoquinodimethane (TCNQ) with four cyano groups and π conjugation bonds have facilitated the formation of organic charge transfer complexes and ion-radical salts like K (TCNQ) and Na (TCNQ) [16][17][18][19][20][21][22]. TCNQ has also been used as a redox mediator for regenerating Oxidoreductase enzymes, especially glucose oxidase, and peroxidase, including the cofactor participating with dehydrogenase enzyme [23][24][25][26][27]. The advantage of the organic redox mediator is low background current even at a higher quantity within graphite paste which in turn allows sensitive and precise probing of various enzymatic reactions [28]. This justified the potential utilization of TCNQ in electrochemical biosensing. Although TCNQ has numerous advantages with electrochemical sensing, the dynamics of electrochemical reaction between biocatalyst and TCNQ is relatively slower as compared to that recorded with ferrocene derivative under similar conditions. This necessitated the search for a novel way to enhance the electrochemical performances of TCNQ as a redox mediator for a variety of practical applications on electrocatalysis, as attempted in the current study.
The redox electrochemistry of the electron transfer mediator within a heterogeneous matrix has been shown to be dependent on the support morphology. The redox electrochemistry becomes sluggish on the exploitation of a nanostructured matrix for redox molecules encapsulation [29]. The sluggish electrochemistry related to the redox mediator encapsulated nanostructured matrix is due to the restricted mobility of the redox couple within the heterogeneous phase. This directed the current study toward a possible approach to fastening the rate of bio-electrochemical interaction between enzyme and redox mediators for reliable and sensitive electrochemical sensing. Herein, an attempt was made to introduce palladium into organically modified silicate (ORMOSIL) precursors as a heterogeneous matrix, involving the active role of palladium chloride [30]. This involved a specific interaction between palladium chloride and precursors of ORMOSIL, i.e., 3-glycidoxypropyltrimethoxysilane (GPTMS) and trimethoxysilane (TMS). GPTMS allowed the reduction of palladium chloride into palladium nanoparticles, followed by triggering the formation of palladium-linked ORMOSIL. While TMS enabled the formation of the Pd-Si-bond within the ORMOSIL matrix. These interactions between precursors of organically modified silica in the presence of ferrocene monocarboxylic acid enabled the formation of palladium-embedded ORMOSIL resulting in excellent redox electrochemistry of ORMOSIL encapsulated ferrocene derivative, and a nanostructured-heterogeneousmatrix behaving as a solid solution during electrochemical biosensing [31,32]. However, it is required to find either analogous variation in the redox electrochemistry of TCNQ that could be recorded as a function of palladium and its nanogeometry, as studied in the current work.
In current work, the use of functional alkoxysilane, especially 3-aminopropyltrimthoxysilane (3-APTMS) which allowed controlled conversion of palladium-positive ions into palladium nanoparticles with cyclohexanone [33] and formaldehyde [34] is demonstrated. The resultant palladium nanoparticles can be calcined at the desired temperature and can be incorporated within graphite paste with TCNQ to reveal the dependence of palladium on the redox electrochemistry of TCNQ. Furthermore, the nanogeometry of palladium nanoparticles can also be controlled simply by altering the amount of 3-APTMS. Therefore, palladium nanoparticles with two different nanogeometry are prepared and employed for incorporation in graphite paste along with TCNQ for finding the dependence of palladium and its nanogeometry on redox electrochemistry of TCNQmodified electrode. The study involvs Transmission electron microscopy, cyclic voltammetry, impedance spectroscopy, and a typical case of ascorbic acid electrochemical sensing for confirming the effect of palladium and its nanogeometry on redox electrochemistry of TCNQ-modified electrode.

Synthesis of PdNPs
The synthesis of PdNPs was done by a previously reported method [33] Briefly, 100 µl ethylene glycol solution of K 2 PdCl 4 (20 mM) was mixed with 5 and 10 µl of 3-APTMS (10 mM) for PdNP-1 and PdNP-2, respectively. The resultant solution was mixed on a cyclo mixture followed by the addition of formaldehyde and further stirring for 10 min. The resultant product was incubated in a microwave for about one minute and centrifuged at 6000 rpm for 10 min to obtain the black color sediment. The sediment was washed two to three times and vacuum oven dried at 80 °C for 2 h.

Fabrication of modified graphite paste electrode
On graphite powder, the newly synthesized PdNP-1 and PdNP-2 were adsorbed. In order to prepare the graphiteblended PdNPs, 100 μl of PdNPs sol was mixed with 100 mg of graphite powder (molecular measure < 20 μM), and the mixture was then ultrasonically processed at 20 kHz for 20 min. The blends were allowed to stand overnight at 50 °C to allow the dissolvable to evaporate completely. To eliminate the organic components, the PdNPs adsorbed on graphite particles were calcined at 600 °C in an N 2 atmosphere. The calcined PdNP-1/ PdNP-2 altogether TCNQ, graphite powder, and binder were homogenized in a processor. Improved carbon paste electrodes (CPE) such as CPE/ TCNQ, CPE/TCNQ PdNP-1, and CPE/TCNQ PdNP-2 systems are produced depending on the presence or absence of PdNPs in the active paste ( Table 1). The paste surface made as discussed was physically polished on clean paper. Bioanalytical Systems provided the electrode frame utilized to construct the carbon paste electrode. An active paste of ingredients has been used to fill the electrode body, as illustrated in Table 1.

Electrochemical measurements
For electrochemical studies, an Electrochemical Workstation Model, CHI 660E Inc., TX, USA, was utilized with a threeelectrode cell with a working volume of 2 ml. As the reference and auxiliary electrodes, a silver/silver chloride electrode (Orion, Beverly, MA, USA) and a Pt electrode were used. The Ag/AgCl electrode is connected to all the potentials indicated below. The working electrode is a modified CPE/TCNQ, CPE/TCNQ-PdNP-1, and CPE/TCNQ-PdNP-2 electrode. The influence of sweep rates on peak current density (j) was investigated by recording cyclic voltammograms

Synthesis and characterization of palladium nanoparticles
The current study revealed the preparation of nanosized palladium particles, i.e., PdNP-1 and PdNP-2 of varying size with different concentrations of 3-APTMS. The XRD spectrum as shown in Fig. 1a

Impact of Pd-nanogeometry on the redox electrochemistry of TCNQ-modified electrodes
After carefully adsorbing PdNP-1 and PdNP-2 on graphite powder, 3-APTMS stabilized nanoparticles were calcined at 600° C to remove all organic parts present on the Palladium nanoparticle. Calcinated nanoparticles were subsequently used to understand the impact of PdNPs on the electrochemistry of TCNQ-modified electrodes as a close contact of PdNPs and TCNQ within graphite paste may facilitate the charge transfer rate during electrochemical measurements. In this regard, interesting findings were recorded. Figure 3 illustrated the results of cyclic voltammetry of (a) CPE/TCNQ, (b) CPE/TCNQ-PdNP-1, and (c) CPE/TCNQ-PdNP-2 within the carbon paste electrode (CPE). Firstly, the variation in the redox performance of TCNQ within the CPE as a response to incorporated calcined PdNP-1 and PdNP-2 was investigated. Figure 3 also showed the cyclic voltammetry of CPE/TCNQ, CPE/TCNQ-PdNP-1, and CPE/TCNQ-PdNP-2 electrodes at different scan rates. The results justified the decrease in anodic (Epa) and cathodic (Epc) peak potentials from 243 to 231 mV. Furthermore, the anodic and cathodic peak currents were steadily raised under similar conditions indicating the reduction of charge transfer resistance as a function of palladium nanogeometry, further confirmed by impedance spectroscopy as in vide infra. Subsequently, the dynamic electrochemistry was investigated based on cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in the absence, and the presence of varying concentrations of ascorbic acid as efficient electroactive species for direct and mediated electrochemistry. The results, as shown in Fig. 4, clearly revealed the significant enhancement of anodic current attributed to the  Differential pulse voltammetry (DPV) of CPE/TCNQ, CPE/TCNQ-PdNP-1, and CPE/TCNQ-PdNP-2 electrodes were subsequently investigated to understand the impact of palladium and its nanogeometry during electrochemical sensing. Figure 5a-c shows the differential pulse voltammograms at varying concentrations of ascorbic acid. The calibration curves made between peak currents of the pulse voltammogram as a function of ascorbic acid are shown in Fig. 5d. A straight-line association was obtained between the differential pulse voltammetry current and AA concentration with a gradual increase in sensitivity of AA sensing and found to be 1.5, 1.9, and 2.5 µA/mM for CPE/TCNQ, CPE/TCNQ-PdNP-1, and CPE/TCNQ-PdNP-2 electrodes, respectively, again confirming the contribution of palladium and its nanogeometry.
The finding recorded in Fig. 5 was further examined on reliable electrochemical sensing of ascorbic acid based on static electrochemistry at a constant potential close to the redox potential of CPE/TCNQ, CPE/TCNQ-PdNP-1, and CPE/TCNQ-PdNP-2 electrodes. Electroanalytical performances of modified electrodes were performed by amperometric measurement under stirring conditions. Amperometric measurement was performed successively by adding ascorbic acid (50-625 µM) to the continuously stirred solution with a working potential of 0.22 V vs Ag/AgCl and recorded. CPE/TCNQ-PdNP-2 showed higher responses than CPE/TCNQ-PdNP-1 and CPE/TCNQ-modified electrodes, as shown in Fig. 6a-c. The standard curve for ascorbic acid (AA) detection by amperometry was constructed (Fig. 6d). The outcomes imply an extensive linear association between the amperometric current and AA concentration. The limit of detection (LOD) was calculated using the following formula.
where S is the standard deviation in the intercept and m is the slope of the calibration curve. The LOD were determined to be 51.61, 44.38, and 30.10 µM for CPE/TCNQ, CPE/ TCNQ-PdNP-1, and CPE/TCNQ-PdNP-2 modified electrodes, respectively. [6] The sensitivity for AA sensing was found to be 0.005, 0.0075, and 0.0148 mA/mM for CPE/ TCNQ, CPE/TCNQ-PdNP-1, and CPE/TCNQ-PdNP-2 electrodes, respectively. This confirms the role of palladium and its nanogeometry on electroanalysis, further confirmed by amperometric responses recorded at varying operating potentials between 100 and 300 mV vs silver/silver chloride (Fig. 6e). This method can be efficiently used for real-time analysis of ascorbic acid in marketed formulations (Supplementary 1). The finding of Figs. 3 and 4 may further be examined to understand the impact of palladium and its nanogeometry based on the change of the current function, which i p /s 1/2 as a function of scan rate, and recorded in Fig. 7a-c. The results on the change of current function on scan rate in all three cases CPE/TCNQ, CPE/TCNQ-PdNP-1, and CPE/TCNQ-PdNP-2 electrodes in the absence of ascorbic acid show a horizontal straight-line, whereas on the addition of ascorbic the current function gradually decreases as an increase in scan rate and finally tend to a straight-line. The current function attains a steady value at a relatively higher scan rate in CPE/TCNQ-PdNP-2 as compared to CPE/TCNQ-PdNP-1 and CPE/TCNQ justifying faster charge transfer dynamics as a function of palladium nanogeometry.

Electrochemical impedance spectroscopy
EIS studies were additionally executed to examine the impact of change on the charge transfer characteristics and recorded for CPE/TCNQ, CPE/TCNQ-PdNP-1, and CPE/TCNQ-PdNP-2 electrodes in 0.1 M phosphate buffer between the frequency range of 1 Hz to 1000 kHz as shown in Fig. 8. A typical Nyquist plot for Randle's circuit has two parts: the semi-circular and the linear element. At low frequencies, the linear element represents the mass transfer diffusion, whereas, at high frequencies, the semicircle's diameter signifies the charge transfer resistance (Rct) [35].
The diameter of the circle component inside the Nyquist plot represents the charge transfer resistance , respectively, which indicates that the maximum charge transfer takes place in the system CPE/TCNQ-PdNP-2. Additionally, with an increase in the concentration of ascorbic acid, the diameter of the circle decreases, and so does the gradual increase in the charge transfer process. The values of charge transfer resistance for CPE/TCNQ, CPE/ TCNQ-PdNP-1, and CPE/TCNQ-PdNP-2 electrodes were calculated as recorded in Table 2, clearly justifying the impact of palladium and its nanogeometry on the redox electrochemistry of TCNQ confirming that an increase in palladium nanogeometry subsequently decreases the charge transfer resistance allowing faster dynamic electrochemistry of TCNQ-modified electrode (Table 3).

Calculation of electrochemical active surface area
The electrochemical active surface area (EASA) of working electrodes was investigated by taking cyclic voltammograms (CVs) of CPE/TCNQ, CPE/TCNQ-PdNP-1, and CPE/TCNQ-PdNP-2 in 0.1 M phosphate buffer at a scan rate of 15 mV, which is shown in Fig. 3a-c, respectively.  Peak current improved with increasing scan rate, and the related linearity graph was found. This represents the anodic and cathodic peak current vs the square root of the scan rate. To determine the EASA of CPE/TCNQ, CPE/ TCNQ-PdNP-1, and CPE/TCNQ-PdNP-2, we utilized the Randles-Sevcik equation [3] described in Eq. (1).
here A denotes the Electrochemical active surface area (cm 2 ), D denotes the diffusion coefficient (cm 2 s −1 ), C 0 denotes the concentration of electroactive analyte, n denotes the number of electrons exchanged during the reversible reaction, Ip denotes peak current (A), and υ denotes the scan rate (Vs −1 ). By putting these values in Eq. (1) The EASA of CPE/TCNQ, CPE/TCNQ-PdNP-1, and CPE/TCNQ-PdNP-2 were calculated to be 0.0547, 0.2350, and 0.2704 cm 2 , respectively. The electrochemical characterizations indicate that CPE/TCNQ-PdNP-2 has superior electrochemical behavior than CPE/TCNQ and CPE/TCNQ-PdNP-1. It is predicted that CPE/TCNQ-PdNP-2 to be a good platform for the electrochemical sensing of ascorbic acid.

Effect of pH
The pH parameter is extremely important for the electrochemical sensing of AA since it strongly influences the oxidation process. Here Fig. 9a shows the graph between current vs pH. This was confirmed by changing the pH of the control sample from 5 to 9 for AA. At pH 7.0 highest current was found thereafter either the pH value increased or the decreased value of the current decreased.

Reproducibility and stability
To determine the sensor's reproducibility, Five TCNQ-PdNPs/CPE-modified electrodes were produced in the same manner. Using these electrodes, anodic peak currents of 0.5 mM AA were measured. This measurement showed a relative standard deviation (RSD) of 2.0%, indicating that the modified electrode made using this procedure is showing good reproducibility. After storing the produced sensor at ambient temperature for 5, 10, and 15 days, the oxidative peak current of AA maintained 98, 96, and 94% of its original value, indicating good stability. As a result, CPE/TCNQ-PdNP-2 is projected to be employed in the detection of AA  with good stability and reproducibility which is shown in Fig. 9b and 9c, respectively.

Conclusions
The current research was undertaken to fasten the dynamic electrochemistry of TCNQ in electrochemical sensing in the presence of palladium in two different nanogeometry. The three different systems, namely CPE/TCNQ, CPE/TCNQ-PdNP-1, and CPE/TCNQ-PdNP-2 electrodes, clearly predict the gradual decrease in charge transfer resistance as a function of palladium nanogeometry. The sluggish electrochemistry of TCNQ-modified electrodes tends to be faster in the presence of palladium, with a gradual increase in terms of peak current as a function of palladium nanogeometry. The TCNQ-modified electrode function was observed to be dependent on palladium nanogeometry even at a faster scan rate. Therefore, palladium and its nanogeometry have a significant effect in redox-mediated electrochemistry along with the introduction of electrocatalysis during electrochemical sensing/biosensing.