Interactive Studies on Synthetic Nanopolymer decorated with Edible Biopolymer and its Selective Electrochemical determination of L-Tyrosine

Herein, an edible biopolymer amine Modified Gum Acacia (MGA), successfully encumbered with Electron Beam irradiated Polypyrrole Nanospheres (EB-PPy NSs), was investigated for the effective role in L-Tyrosine (Tyr) biosensing application. The morphology of EB-PPy NSs decorated MGA (EB-PPy/MGA) hybrid nanobiocomposite has been studied by Scanning electron microscopy and its affirmed interactions were characterized by X-ray diffraction, Raman, FT-IR spectroscopy, UV-Visible spectroscopy, Thermo Gravimetric Analysis and Vibrating Sample Magnetometer. The hybrid nanobiocomposite manifested diamagnetic behavior with reduced saturation magnetization (Ms = 1.412 × 10−4 emu/g) to produce more adhesive surface. Amine chains in EB-PPy NSs and hydroxyl groups of MGA contributed to effective immobilization, thus enabling suitable orientation for Tyr determination. The electrochemical analysis illustrated that the proposed nanobiocomposite based sensor exhibited an excellent electrocatalytic activity toward selective determination of Tyr in the linear range of 0.4 to 600 µM with a lower detection limit of 85 nM, low oxidation potential of 0.72 V and good selectivity. Finally, the reliability of the constructed EB-PPy/MGA for Tyr detection was demonstrated in real samples.

www.nature.com/scientificreports www.nature.com/scientificreports/ EB-PPy NSs with decreased intensity compared to pristine PPy (Fig. S5(B)). Hong et al. reported the influence of EB irradiation on PPy results in increased intensity of main characteristic peak due to the structural defects leads to shortening of π-conjugation length 28 . Here, we observed the decrease in intensity of EB irradiation that informs lengthening of π-conjugation length of PPy NSs and confirmed occurrence of crosslinking. Subsequent decrease in intensity was observed in EB-PPy/MGA due to the presence of MGA and further evidenced its promising interaction.  The FT-IR spectroscopy of as-prepared samples was presented in Fig. 5(A) confirms the changes of amine and alkyl groups in cross linked EB-PPy NSs and MGA for composite formation. In that, it is observed main characteristic peaks of EB-PPy NSs (curve b) shows various vibrational frequencies at 1529 cm −1 (C=C stretching), 1419 cm −1 (C-N stretching), 1300 cm −1 (=C-H in-plane deformation) of pyrrole ring 29 . The main characteristic peaks for GA and modification of -OH groups to -NH 2 group were explained and supported in Fig. S6(A). The presence of EB-PPy NSs and MGA in nanobiocomposite also evidenced by the observation of individual bands with sharper peak at 3435 cm −1 in the spectra and increase in peak intensities ascribed the reduced OH groups in MGA due to successful adsorption with EB-PPy NSs.
This hypothesis further gets confirmed by UV-visible absorption spectra of MGA, EB-PPy NSs and EB-PPy/ MGA samples (Fig. 5(B)). The observation of crosslinking in EB-PPy NSs further supported and explained in Fig. S6(B). In Fig. 5(B), the curve 'a' indicates the absorption peaks at 270 nm, 450 nm corresponds to terpyrrole oligomers and π-π* transition of high molecular weight PPy 30 . From curve 'c' , it could be observed that the increase in intensity and slight shift of absorption band at 290 nm towards higher wavelength ascribing the less terpyrrole oligomers in EB-PPy/MGA. The decrease in intensity of absorption band resulted due to the proportional addition of EB-PPy NSs into MGA surface and also affirms the strong interaction.
To further elucidate the thermal stability of the prepared nanobiocomposite, the Thermo Gravimetric Analysis (TGA) was performed for the EB-PPy NSs, MGA and EB-PPy/MGA samples. From Fig. 5(C), the curve 'a' exhibits five steps of weight loss starting from 154 °C, 204 °C, 429 °C, 596 °C and 859 °C respectively. In amine treated GA, the first weight loss occurred at 90 °C due to the presence of significant amount of moisture in sample and the second weight loss at 297 °C to 334 °C 31 . In EB-PPy/MGA sample, the observed five steps of weight loss starts from 145 °C, 306 °C, 411 °C, 507 °C and 840 °C (curve c). The obtained residual weight% at 900 °C for EB-PPy NSs, MGA and EB-PPy/MGA were 44.14%, 32.27% and 32.7% from the total sample weight of 5.123 mg, 14.28 mg and 2.148 mg respectively. Thus, this result attributed the significant increase in thermal stability of MGA while embedding with EB-PPy NSs and confirms the strong affinity towards the polymer binding sites. Here the prepared edible biopolymer with synthetic polymer based hybrid composite obtained the total weight loss of 70% at 600 °C while 75% at 900 °C, suggesting the improvised thermal stability behavior in the hybrid composite. Thus the prepared novel hybrid composite demonstrated significant enhanced thermal stability over biopolymer based composite which gives new insight in development of polysaccharide based composite by making possible interaction with inorganic nanomaterials. www.nature.com/scientificreports www.nature.com/scientificreports/ Magnetic measurements of EB-PPy NSs, MGA and EB-PPy/MGA nanobiocomposite were characterized using Vibrating Sample Magnetometer (VSM) and presented in Fig. 5(D). It has shown negative magnetic moment which denotes diamagnetic nature with observed saturation magnetization (M s ) of 1.571 × 10 −4 , 1.299 × 10 −4 , 1.412 × 10 −4 emu/g. The decrease in M s value indicates the grain size reduction when compared to bulk material 32 . Here, we found the reduction of M s in EB-PPy/MGA as compared to EB-PPy NSs due to the increased surface area. So the diamagnetic behavior of the prepared hybrid nanobiocomposite could be applicable as superconducting medium and tuning of magnetic behavior also possible by incorporating paramagnetic or ferromagnetic materials for the separation of small molecules 33 . Therefore, the reduced grain size of EB-PPy with MGA confirms the strong steric interaction in proposed nanobiocomposite and thus creating more active sites for Tyr sensing. Well defined reversible voltammograms with E 1/2 value of 0.202-0.265 V were obtained. The gradual decrease in peak-to-peak separation (ΔE p : 70 mV) of EB-PPy/MGA modified GCE compared to other modified GCE represents a high reversibility of the nanobiocomposite due to the increased total number of -OH groups on the composite introducing negative charges on their surface, which in turn interacts with Fe 3+ at the oxidation potential 0.22 V. Moreover, the voltammetric response of EB-PPy/MGA/GCE was found highly reversible from reduced i pa /i pc ratio ( Table 1). The influence of charge transfer mechanism in PPy NSs at different intensities (10 kGy, 20 kGy and 30 kGy) of EB irradiation was supported and explained in Fig. S1 and Table S1. The effect of different scan rate on EB-PPy/MGA and its linear fit of anodic and cathodic peaks were reported and explained in Fig. S7 using 1 mM of [Fe(CN) 6 ] 3-4in 0.1 M KCl as buffer. Further to elucidate the improvisation in electron transfer, the rate constant k 0 was calculated by following standard rate constant relationship in Eq. (1) 34 . The rate constant value of EB-PPy NSs was better than pristine PPy NSs depicts the changes in structural as well as electron transfer properties. As can be seen from Table 1, a gradual increase in i pa , k 0 and gradual decrease in ΔE p values for EB-PPy/MGA/GCE, independent of scan rate clearly indicate the facile electron transfer reaction. www.nature.com/scientificreports www.nature.com/scientificreports/

Electrochemistry of EB-PPy/MGA.
The charge transfer kinetics of the bare, MGA, EB-PPy NSs, EB-PPy/MGA were measured by Elecrochemical Impedance Spectroscopy (EIS) in the frequency region from 100 kHz to 1 Hz and the DC potential 250 mV and AC potential 250 mV in the presence of 1 mM of [Fe(CN) 6 ] 3-4in 0.1 M KCl as redox probe. The Nyquist plot and the Randle's equivalent circuit used to fit the experimental EIS curves for different modified electrodes were recorded in the Fig. 6(B). The equivalent circuit comprising of Rs -solution resistance, R ct -charge transfer resistance, W -Warburg element, Q -Constant Phase Element (CPE), R f and C f -resistance and capacitance of another layer developed after composite interaction 35 . As compared to earlier reported circuits 36 , this circuit (R(Q(RW))(CR) resulted well fitting which shows specified performance of the as-prepared hybrid composite. In this, CPE was used instead of C dl due to the inhomogeneity of electrode surface 26 and the corresponding additional layer can be evaluated using R f and C f components. Interestingly, it is observed from Table S1, the charge transfer resistance on 20 kGy EB-PPy NSs was ~13 times lower than that of pristine PPy NSs implying the facile electron transfer kinetics resulted from intermolecular crosslinking behavior in polymer backbone. The consecutive decrease in the diameter of the high frequency semicircle of EB-PPy/MGA/GCE in Fig. 6 Figure S8 shows the maximum oxidation peak current of Tyr obtained at pH 7 and decreased oxidation peak current at other pH values. In addition, the oxidation peak potential of Tyr shifts negatively with an increase in pH due to the effective involvement of protons in electrode reaction process. The electrochemical behaviour of Tyr for bare, EB-PPy NSs, MGA, EB-PPy/MGA/GCE were investigated by using CV at a scan rate of 50 mV s −1 and reported in Fig. 6(C). It can be observed that the oxidation peak current for EB-PPy/MGA/GCE found increased, compared to other modified electrodes due to the electrosteric stabilization of EB-PPy NSs by MGA on electrode surface that facilitates enhanced electrocatalytic behavior towards Tyr. The effect of scan rate on electrochemical behavior of Tyr for the EB-PPy/MGA/GCE was investigated in 0.1 M PBS and plotted in the Fig. 6(D). As increase in scan rate, a gradual increase in oxidation peak currents of Tyr was noticed and also found linear relationship between peak current and square root of scan rate in the range of 10 to 100 mV s −1 with linear equation y = 18.944x + 7.1380 and R 2 = 0.9848. This study supports the diffusion controlled phenomena of EB-PPy/MGA modified electrode.

Nonenzymatic electrocatalytic detection of Tyr. Under optimized conditions, the determination of
Tyr for EB-PPy/MGA nanobiocomposite was carried out in 0.1 M PBS by Square Wave Voltammetry (SWV) and the results are illustrated in Fig. 7(A). It can be seen that the increasing concentration of Tyr results linearly  www.nature.com/scientificreports www.nature.com/scientificreports/ increase in SWV anodic peak currents with lower detection limit of 85 nM and linear range of 0.4-600 µM. The corresponding linear regression equation is I (µA) = 6.2529 + 1.4906C (µM) (R 2 = 0.9994) for Tyr (Fig. 7(B)). The calibration curve for higher concentration of Tyr also have shown dual linear relationship of increase in oxidative peak current and supported in Fig. S9. The -NH 2 group modified glycoprotein in GA responsible for electrostatic interaction towards Tyr, whereas the net amount of negatively charged polysaccharides in GA provide strong electrostatic and electrosteric interaction towards EB-PPy NSs. Thus, the synergistic effect in prepared hybrid nanobiocompsite enhanced the electrocatalytic behavior towards Tyr with better stability. The overall possible reaction mechanism is illustrated in Fig. 8. Table 2 shows a comparison of the prepared EB-PPy/MGA with some of the reported hybrid composite modified electrodes for Tyr sensing and found that the nanobiocomposite exhibited lowest oxidation potential (0.72 V) of Tyr sensing with low detection limit and wide linear range.

Stability, reproducibility, Anti-Interference Property and Real sample detection of EB-PPy/ MGA/GCE. A selective determination of Tyr in the presence of possible interfering foreign compounds using
EB-PPy/MGA/GCE was studied and shown in Fig. 9(A). The oxidation current of Tyr remain almost unchanged, even in the presence of 100-fold increase in physiological interfering compounds such as L-phenylalanine, L-cysteine, L-Arginine, L-histidine, L-aspartic acid, glycine, uric acid, dopamine (10 mM). Thus EB-PPy/MGA   www.nature.com/scientificreports www.nature.com/scientificreports/ nanobiocomposite was highly selective towards the determination of Tyr even in the presence of interfering compounds.
The stability of the EB-PPy/MGA nanobiocomposite was studied in SWV of 100 cycles with 200 µM of Tyr in 0.1 M PBS and shown in Fig. 9(B). For first 5 cycles there is no significant change in anodic peak current and then it slowly decreases as 4.49%, 5.51%, 6.1% at 20, 50, 100 cycles respectively. The reliability of the prepared nanobiocomposite was observed from EB-PPy/MGA modified 10 different electrodes with 50 µM of Tyr and the calculated RSD was 3.5% respectively. To validate the proposed analysis method, the detection of Tyr content in real samples such as chicken meat and cow milk were analyzed with EB-PPy/MGA/GCE by standard addition method and significant amount of Tyr was found (Fig. S10). The accuracy of Tyr content detection for the designed EB-PPy/MGA/GCE in real samples were performed by SWV studies using Tyr spiked samples of grinded chicken, milk and human urine samples. The concentration of Tyr in real samples were found with satisfactory recovery% and listed in Table 3. Therefore, the proposed EB-PPy/MGA nanobiocomposite can be a promising platform to determine Tyr in real samples.

conclusions
In Summary, we have successfully developed the electrosterically encumbered EB-PPy/MGA hybrid nanobiocomposite and also amended from various characterization techniques. The increase in adhesive interfaces of EB-PPy with MGA through hydrophilic/hydrophobic and electrostatic interactions enriched more active sites in EB-PPy/MGA nanobiocomposite facilitating the biosensing behavior of Tyr. The fabricated EB-PPy/MGA based electrochemical biosensor has shown high sensitivity, selectivity and stability for the selective determination of Tyr with lowest detection limit of 85 nM. Moreover, the EB-PPy/MGA hybrid composite exhibited diamagnetic behavior with reduced grain size which facilitates more surface area and thus providing enhanced sensitivity. The versatility of the fabricated biosensor is also proven from the qualitative detection of Tyr content in commercial food products and human urine samples thus it can be effectively used for future clinical diagnosis application. Preparation of PPy NSs and electron beam irradiation. Nanostructured PPy was prepared by oxidative polymerization of pyrrole monomer using FeCl 3 at maintained temperature of −5 °C and details are published in our earlier reported procedure 29 . The obtained black colour powdered form of PPy NSs were irradiated   www.nature.com/scientificreports www.nature.com/scientificreports/ with 8 MeV EB with different dosages 10 kGy, 20 kGy and 30 kGy for the duration of 4 h, 6 h and 8 h respectively. Since 20 kGy EB-PPy NSs has shown good redox behavior (Fig. S1), it is chosen for the experimental studies.

Materials. Reagent grade
Preparation of MGA. An aqueous solution of GA was amine modified by the addition of ethylenediamine (C 2 H 8 N 2 ), responsible for the substitution of -OH group with NH 2 CH 2 CH 2 NH 2 and then reduced to -NH 2 group 37 . In same way, 1 g of GA powder was dissolved in 250 mL of de-ionized water with stirring. In that 15 mL of C 2 H 8 N 2 was added which substituted the primary -OH groups of galactose and -COOH groups of glucoronic acid in GA with -NH 2 CH 2 CH 2 NH 2 and kept for overnight stirring. The reaction mixture was then added with 1 M (50 mL) HCl for the reduction of -NH 2 CH 2 CH 2 NH 2 into -NH 2 and stirred for 3 h. After that, the product was separated and washed several times with acetone to remove -CH 2 CH 2 NH 2 compounds. The amine modified polymer product was then dried in freeze-dryer.
Preparation of EB-PPy/MGA modified GCE. Prior to the modification in Glassy Carbon Electrode (GCE), it was polished successively using 1.0, 0.3 0.05 µm of α-alumina powder, washed using de-ionized water, and finally rinsed thoroughly with ethanol. For the selection of appropriate composite proportion, different weight ratio of EB-PPy NSs:MGA was studied from Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS) (Fig. S2) and the ratio of 1:1 has shown higher oxidation peak which is clinched for the paper work. The mixture of EB-PPy/MGA was dispersed in 1 mL de-ionized water with ultrasonic treatment for 6 h and 10 µL of this homogeneous suspension (5 mg mL −1 ) was drop casted onto GCE and then allowed to dry at room temperature for 2 h, resulted the EB-PPy/MGA modified GCE. For comparison purposes, 10 µL of the homogeneous suspension (5 mg mL −1 ) of EB-PPy NSs, MGA were also separately coated on bare GCE to obtain individual modified electrodes.
Preparation of real samples. The real samples for Tyr detection was prepared according to reported literature 38 . The sample preparation procedure for the chicken was carried as follows: 0.5, 1 and 2 g of grinded chicken sample and dissolved in 20 mL de-ionized water and were mixed with 0.5, 1 and 1.5 mL of TCA to precipitate proteinous components in the prepared samples. Similarly, the milk sample preparation was carried as follows: 1, 2 and 10 mL of milk sample were mixed with 0.2, 0.4, 2 mL of TCA to precipitate proteinous components. The mixture of samples were kept in vortex mixer for 1 min and centrifuged at 4000 rpm for 15 min separately. The supernatant were transferred to another centrifuge tube and filtrated by 0.45 µm syringe filter. The collected filtrate samples of chicken and milk samples were diluted ten times and twice with 0.1 M PBS respectively for further analysis. The human urine samples from 2 healthy volunteers were centrifuged at 4000 rpm and filtrated and then the filtrate sample diluted twice with 0.1 M PBS. All the prepared real sample solutions were adjusted to pH 7.0.
Characterization. The electrochemical measurements were prepared with a CHI6005D electrochemical workstation (Austin, USA). A conventional three-electrode system was used for all electrochemical experiments, which consisted of a platinum wire as counter electrode, an Ag/AgCl/3 M KCl as reference electrode, and glassy carbon electrode (0.07 cm −2 ) as working electrode. The electrochemical reaction was carried out in PBS at pH 7. The EB irradiation on PPy NSs was done by 8 MeV Microtron at Mangalore University. The Scanning Electron Microscopy (SEM) images for PPy, EB-PPy, MGA and nanobiocomposite were recorded using Zeiss operating at 21.00 kV. The High resolution Transmission Electron Microscopy (HR-TEM) images of PPy, EB-PPy and EB-PPy/MGA samples were recorded using JEOL-2100 operating at 200 kV. The XRD patterns were carried out using Bruker Germany D8 advance instrument (λ = 1.5418 Å) operated at 30 mA, 40 kV and using Cu kα radiation. The Raman spectrum was obtained using an imaging spectrograph (model STR500 mm focal length) laser Raman spectrometer (SEKI Japan).