Green Synthesis of Graphene Based Biomaterial Using Fenugreek

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Green Synthesis of Graphene Based Biomaterial using Fenugreek Seeds for Lipid Detection Chandan Singh, Azahar Ali, and Gajjala Sumana ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.5b00923 • Publication Date (Web): 15 Jan 2016 Downloaded from http://pubs.acs.org on January 20, 2016

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ACS Sustainable Chemistry & Engineering

Green Synthesis of Graphene Based Biomaterial using Fenugreek Seeds for Lipid Detection Chandan Singh1,2, Md. Azahar Ali1, GajjalaSumana1*

1

Biomedical Instrumentation Section, CSIR-National Physical Laboratory, Dr. K. S. Krishnan Marg,

New Delhi, India 110012. 2

Academy of Scientific and Innovative Research (AcSIR), CSIR-National Physical Laboratory

Campus, Dr. K.S.Krisnan Marg, New Delhi, India 110012.

ABSTRACT A novel, green and sustainable route has been introduced for biosynthesis of palladium nanoparticles (PdNPs) anchored reduced graphene oxide (rGO) platform for protein conjugation and detection of triglycerides. Fenugreek seeds (FS) were utilized for the simultaneous green reduction of Pd salt and graphene oxide (GO), which also act as stabilizing and decorating agent for in situ grown PdNPs on rGO sheets. The well dispersed, homogeneous and biologically reduced PdNPs having average size of 3.5 nm on rGO sheets are electrophoretically deposited on indium tin oxide (ITO) glass substrate. The biosynthesized Pd-rGO sheets were characterized using spectroscopic, electrochemical techniques and further utilized for co-immobilization of lipase and glycerol dehydrogenase (LIP-GLDH) enzymes using EDC-NHS coupling chemistry. The LIP-GLDH conjugated PdrGO platform was used for electrochemical detection of triglycerides. The green and nontoxic approach offers enhanced stability of the fabricated biosensor as phytochemicals (present in FS extract) residues available on the surface of Pd-rGO nanocomposite might have prevented the loss in the structure of the immobilized biomolecules/enzymes by

ACS Paragon Plus Environment


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providing biocompatible coating. The fabricated Pd-rGO based biosensor shows a wide detection range of 25 - 400 mgdL-1 with an excellent sensitivity of 1.3 µA mgdL-1cm-1.

Keywords: Green Chemistry, Graphene oxide, Palladium nanoparticles, Biosensors, Lipid detection, Fenugreek Seeds, Sustainability,

*Corresponding

author:

[email protected];

Phone:

+91(011)45609152.


+91(011)45602439,

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INTRODUCTION Since last two decades, green chemistry has provided innovative tools to reduce the application of toxic and hazardous chemicals in both research and industry.1Utilization of these chemicals in the field of material synthesis is very frequent but recently green chemistry has replaced them with eco-friendly and sustainable methods.2 Green chemistry offers controlled, low temperature and biocompatible route to synthesize nanostructured materials of metal, metal oxide and carbon including graphene. Graphene has been considered as twodimensional (2-D) atomic crystal and building blocks of graphene based nanocomposites have enormous applications for the development of health-care devices. There has been potential interest in GO sheets due to various oxygen functional moieties present on its surface which has been utilized for the development of nanoelctronics devices, field effect transistor-based sensors, point-of-care biosensors (POC), and bio-imaging3, 4. GO is considered as electrically insulating material due to its disrupted sp2 bonding network and can be restored by performing the reduction of GO5, 6.In rGO, the 2D crystal of sp2 hybridized carbon provides exciting electronic properties such as room-temperature quantum Hall effect and mass less Dirac fermions7-9. Due to their extra ordinary electronic properties, rGO sheets are strong candidates for advanced nanoelectronic devices such as gas sensors, transistors and integrated ballistic carrier devices based on nano-patterned epitaxial graphene10-12. In addition, rGO also contains different oxygen vacancies, which can be utilized for the attachment of biomolecules making rGO highly suitable candidate for the next generation POC devices13. Reduction of GO results into the partial restoration of graphitic network, conventionally achieved using chemical, thermal and electrochemical pathways. For chemical reduction,various chemicals such as hydrazine (N2H4) and sodium borohydride (NaBH4) have widely been explored14-16.In particular, hydrazine has several disadvantages,



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for example, during removal of oxygen functional groups nitrogen tends to attach with GO via covalent attachment and tend to form hydrazones, amines, aziridinesetc14. These C-N residuals functioning as n-type dopants have profound influence on the electronic structure of chemically derived rGO17. These sheets have higher sheet resistance creating a barrier in its applications. 12, 13However,there is no simple procedure available to remove these impurities, which may be present in C: N ratio of 16.1:1 as reported by Stankovich et al.18.Chemically derived rGO also suffers the problem of low solubility and stability in water and organic solvents and due to π-π stacking, they tend to form irreversible agglomerates19. Further, these chemical modification induces toxicity, for instance, hydrazine derived reduced graphene sheets contains toxic C-N groups introduced during the reduction process20,

21

.Thus, the

toxicity of chemically modified rGO sheets perhaps unsuitable for various biomedical applications. Thermally mediated reduction of GO leaves behind topological defects and vacancies which influences the electronic properties resulting in the decrease of the ballistic transport path length and introduces scattering sites22.Although, the electrochemical reduction of GO can avoid the use of hazardous chemicals, however it perhaps suffers the problem of scalability since the deposition of rGO onto conductive electrode. In this context, implementation of greener reduction approach can provide a sustainable alternative approach for mass production of rGO with non-toxic property and favourable environment for biomolecules. Fenugreek (Trigonella foenum-graecum) is mainly a spice crop belong to the family of Fabaceae. It is a semi-arid plant and cultivated worldwide. Its pods contain 10-20 seeds having cuboid-shaped, yellow-to-amber colored are used to prepare extracts or powders mainly for cuisines and medicinal uses. FS contain various phytochemicals such as sotolone, eugenol, caproic acid, butanoic acid etc. as shown in Figure 1and have been utilized as reducing and stabilizing agent for synthesis of various nanoparticles including gold, silver and selenium23-25.Paul Anastas and John Warner have defined the principles of green


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chemistry which includes prevention of hazardous waste, designing of less hazardous chemical in synthesis process, design of safer chemical and products, use of renewable feedstock, designing chemicals and products to degrade after use and minimize the potential for accidents.26The application of FS extract for the reduction of GO and metal salts offers a non-toxic route and avoids the generation of hazardous waste and promotes the use of renewable feedstock like spices. Thus, green chemistry plays an important role in

the

sustainable synthesis and may be termed as “ideal synthesis” as it is based on green, nontoxic and eco-friendly chemicals27, 28.Various leaf, root and seeds of plants were utilized for the reduction of metal salts to produce nanoparticles29.Researchers have explored different plant extracts for the reduction of GO, for example, an aqueous extracts of Colocasia esculenta, Mesua ferrea Linn. and peel extract of orange (Citrus sinensis) have utilized for reduction of GO30.Theleaf extract may contain the phytochemicals which can easily be converted into particular quinines via oxidation. The presence of multiple pyrogallol and catechol moieties of polyphenolic compounds in green tea have been utilized for the efficient reduction of GO31,

32

.An aqueous extract of carrot root has also been explored for fast

reduction of GO at low temperature33. Simultaneous reduction of both metal salt and GO resulting in nanoparticles decorated rGO sheets using plant extracts can provide an ecofriendly, sustainable and cost-effective route. Due to large surface area, chemical stability and low cost, GO can be considered as a supporting material for the growth of metal nanoparticles34-37. The availability of various oxygen functionalities at GO surface can play an important role fornucleation and growth of metal nanoparticles. PdNPs on GO sheets are widely used in heterogeneous catalysis as it creates reproducible ohmic contacts with GO38,

39

. Theoretically, the interaction of metal

nanoparticles and graphene has been investigated using density functional theory (DFT) which helps in determining the position of foreign atom on the hexagonal graphene sheet40, 41. On the basis of adsorption energy and structural properties, three possible sites are B (top of



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C-C bond), H (centre of hexagon) and T (top of carbon atom) have been predicted for absorption ofmetal atoms as shown in Figure 242. According to DFT theory, site B has been predicted as the most favourable site for adsorption of Pd atom on graphene, although Pd has filled 4d orbitals, it strongly hybridize with graphene states implying almost a covalent bonding37. Khomyakov et al. have explained the generation of additional interaction states and

transmission

channels

between

PdNPs

and

GO

leading

to

the

strong

bonding37.Integration of metal nanoparticles with rGO sheets can prevent the aggregation, since acting as spacer, metal nanoparticles increase the distance between rGO sheets43.This fascinating interaction chemistry proves GO a suitable platform for growth and anchoring of PdNPsthat can significantly improves the sensitivity and detection limit of a biosensor. In this manuscript, we have demonstrated the reduction of Pd salt and GO sheets simultaneously using FS extract. FS acts as a reducing agent for simultaneous growth of PdNPs on rGO sheets and further it has also acted as stabilizing and decorating agent for homogenously distributed PdNPs on rGO sheets. Biosynthesized Pd-rGO composite was characterized by various spectroscopic and microscopic techniques. The available functional groups (–COOH) on Pd-rGO surface has been utilized to functionalized with –NH2group of bienzyme (LIP-GLDH) via covalent strong interactions. This bienzyme conjugated Pd-rGO on ITO electrode has been explored for detection of triglycerides via cyclic voltammetric technique (CV). EXPERIMENTAL SECTION Chemical and Instruments Graphite powder flakes (45 µm, >99.99 wt%) were purchased from Sigma Aldrich, USA. Palladium chloride salt (PdCl2) was purchased from SD Fine Chemical, Mumbai, India. LIP with specific activity of 48 Umg-1 and GLDH with specific activity of 77 Umg-1 have been


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procured from Fluka. Nicotinamide adenine dinucleotide (NADH), N-hydroxysuccinimide (NHS), N-ethyl-N-(3-dimethylaminopropyl carbodiimide) (EDC), and trybutyrin (TB), have been purchased from Sigma-Aldrich, USA. FS were purchased from local market in New Delhi, India. Deionized water (DI, Millipore) was used to make a phosphate buffer saline containing 50 mMNaCl of pH 7.4. High resolution transmission electron microscopy (HR-TEM) and selected area electron diffraction (SAED) pattern have been utilized to characterize rGO, PdNPs and Pd-rGO nanocomposite (Tecnaii-G2F30 STWIN). The morphological observation of Pd-rGO nanocomposite before and after enzyme co-immobilization has been confirmed by scanning electron microscopy (Hitachi S-4800 FE-SEM) at an acceleration voltage of 10 kV. UVvisible and FT-IR studies were performed structural properties and growth on PdNPs on rGO sheets using Perkin-Elmer spectrophotometer and Perkin-Elmer, Model 2000, respectively. The Raman studies have been conducted to investigate the reduction of GO using Raman Spectroscopy (HR800 LabRam, Horiba/Jobin-Yvon), in an excitation wavelength of 532 nm at room temperature. Atomic force microscopy (AFM) has been carried out to estimate the height profile of rGO using multimode analysis (Bruker).The electrochemical analysis have been performed using an Electrochemical Analyzer (AUT-84275) in phosphate buffered saline (PBS, pH 7.4) containing 5 mM [Fe(CN)6]3-/4- as a redox probe. Synthesis of GO

GO has been synthesized as described in previous reported literature 45. In brief, combination of concentrated H2SO4 to H3PO4, (9: 1, 240 ml/26.7 ml) has been mixed with 2 gms of graphite flakes and 12 gms of KMnO4. Due to exothermic nature of this reaction KMnO4 was added with a time span of 15 min to avoid explosion. Further, the reaction mixture has been kept under magnetic stirring condition for 12 hrs at a constant temperature of 50° C. Subsequently, this reaction was quenched by the addition of 300 mL of ice with 2.5 ml of



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30% H2O2. Finally obtained yellow coloured slurry mixture was centrifuged, filtered followed by washing with 30% HCl and then washed with distilled water subsequently until the pH ~7.0. This mixture was dried at 65 °C to obtain solid dry powder of GO. Preparation of FS extract For the preparation of extract 10 gms of FS was washed several times with DI to remove various impurities and then immersed into 50 mLof DI water for 12 hrs at room temperature. This solution was filtered using filter paper (Whatman Grade No. 40) and then centrifuged at 8,000 rpm for 15 minutes and supernatant was collected and stored at 4°C for further use. Synthesis of Pd-rGO nanocomposite For Pd-rGO nanocomposite, 5 mgs of PdCl2 was added to 15 mL of a GO aqueous suspension (1.0 mg/mL) and the resulting solution was aged for 30 min at 50°C to allow the interaction of palladium ions with the dispersed GO surface. Again, drop-wise addition of 5 mL of FS extract into the prepared solution at 80°C with constant stirring for 18 hrswhich provides the simultaneous reduction of both GO and Pd (II) complex. For reduction, 5 mL of FS extract has been added into 20 mL of GO (1.0 mg/mL) and kept at 80°C on magnetic stirrer for 18 hrs. Fabrication of Pd-rGO onto ITO electrode Electrophoretic deposition (EPD) technique was utilized for deposition of GO and Pd-rGO composite. Electrophoretically depositedgraphene films provide high density and uniform thickness with numerous edges normal to the film surface

46

. The colloidal solution of Pd-

rGO was deposited onto ITO (sheet resistance of 30 Ω cm−1) using two electrode cell and applying DC voltage (50 V) for 2 min in acetonitrile solution. Further, 10−5–10−4mol of Mg (NO3)2.6H2O was added into the suspension as an electrolyte for EPD. In this electrochemical system, a platinum (Pt) foil (1cm×2cm) acts as the cathode and a pre-cleaned ITO-coated


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glass substrate as anode separated by 1.0 cm. After deposition, the electrodes were removed from the suspensions and washed with DI followed by drying.

Fabrication of biosensing platform Before enzyme co-immobilization, 1:1 solution of EDC (0.4 M) and NHS (0.1 M) was spread uniformly onto the electrophoretically deposited Pd-rGO surface and kept in a humid chamber for about 4 hrs. The EDC-NHS coupling chemistry activates –COOH groups present in Pd-rGO surface. 20 µL of LIP and GLDH in 1:1 ratio was drop casted on the surface of Pd-rGO for co-immobilization. –NH2 groups in enzyme molecules form covalent strong bond (C-N) with –COOH groups in Pd-rGO surface. Thus, fabricated bioelectrode was then washed with phosphate buffer to remove the unbound enzymes and stored at 4ºC when not in use. RESULTS AND DISCUSSION Raman Spectroscopic analysis Raman spectroscopy has been considered as a powerful tool to distinguish between GO and rGO since it can easily examine number of layers, defects and electronic structure. In general analysis is based on the G band occurring due to the first order scattering of the E2g phonon of sp2 C atoms typically observed at 1580 cm-1and the D band due to a breathing mode of kpoint photons of A1g symmetry which can observed at 1350 cm-147, 48. A significant increase in the intensity of D band was observed after reduction of GO due to introduction of defects. In the present study, the intensity of ID/IG for GO was estimated as 0.90, whereas after reduction the ratio was shifted to 0.98 (Figure 4a). This increase in ID/IG is mainly due to existence of unrepaired defects present after the elimination of negatively charged oxygen moieties of GO. In addition to D and G bands, a 2D peak (at 2697 cm-1)has been observed, which indicates the presence of multilayered graphene sheets48, 49.Raman spectra can also use for determination of average crystallite size (La).Thus, the average crystallite sizes were



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calculated as 18.7 nm and 17.1 nm for GO and rGO, respectively, using Eq.150.The decrease in La may be due to change in the density of defects during the reduction process.

L nm = 2.4 × 10 λ

(1)

whereλ is the wavelength of the laser in nm, while ID and IG are the intensities of D and G bands, respectively. UV-visible spectroscopic studies GO is considered as extremely favourable matrix for PdNPs due to the presence of various oxygen moieties (carbonyl, carboxylic, hydroxyl and others) on its surface as evident by FTIR studies. These oxygen groups help for the nucleation of PdNPs which provide an overall negatively charged surface. This may lead to strong electrostatic interaction between Pd2+ and GO surface. During synthesis of Pd-rGO, initially, Pd2+-GO solution was kept for 30 min to achieve electrostatic interactions among them. On addition of FS extract into Pd2+-GO mixture solution, the subsequent reduction of Pd (II) enabling the growth of PdNPs on GO sheets and simultaneous conversion of GO to rGO which was confirmed by UV-visible studies. GO shows a peak at 230 nm arising due to π-π* transitions of aromatic C-C bonds. After reduction of GO, this peak is shifted to 270 nm with increasing absorbance (Figure 4b). Both changes indicate the restoration of electronic conjugation upto some extent within carbon framework of graphene sheets after the reduction (curve b)51, 52. Further, curve (c) show the mixer of the GO and Pd(II) salt solution clearly demonstrates a peak at 230 nm indicating the presence of unreduced GO. Two peaks at 310 nm and 420 nm are due to ligand-to-metal charge-transfer transition of the Pd(II) ions present in the solution

53

. After

the simultaneous reduction of Pd2+ ions and GO, the peak of GO is shifted to 270 nm and two peaks at 310 nm and 420 nm are found to disappear indicating the formation of PdNPs on rGO sheets(curve d)54. Disappearance of the absorption peaks is also supported by theoretical studies of surface plasmon resonance absorption of PdNPs as discussed in previous


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literature46. FS extract shows two intense peaks at 270 nm and 335 nm (curve e)due to the presence of various flavoinds such assotolone, caproic acid and polyphenols (eugenol, linalool). FT-IR Spectroscopic characterization To confirm the conversion of GO to rGO and further in situ simultaneous reduction of PdrGO nanocomposite, FT-IR studies have been performed (Figure 5). In GO, the characteristic bands seen at 1610 and 1409 cm-1 correspond to C=C stretching and O-H bending of the carboxyl groups. Additionally, a small peak seen at 1700 cm-1 attributed to the C=O stretching of GO. Peak seen at 1130 cm-1 assigns to C-O stretching. After the reduction of GO, the intensities of all characteristic peaks are found to reduce compared to GO. Peak centred at 1610 cm-1 become broadened may be due to the absorption of phytochemicals present in FS extract. In addition, FS extract shows other peaks at 1415 and 1140 cm-1 may be assigned to various components such as sotolone (C6H8O3), eugenol (C10H12O2 ), linalool (C10H18O ), butanoic acid (C4H8O2) and caproic acid (C6H12O2) etc. A broad peak has been appeared at 1050 cm-1 for Pd-rGO nanocomposite due to C-O stretching bending. In the case of Pd-rGO nanocomposite, a broadened peak centred at 1610 cm-1 has been found which is similar to rGO, while the intensities of other peaks have been reduced due to attachment of PdNPs. Morphological studies HR-TEM imagesof rGO and PdNPs decorated rGO are shown in Figure 6. Image (A) shows the overview of the wrinkled and folded rGO flakes. Inset shows the SAED pattern of rGO, where two diffraction graphitic planes (002) and (004) have been observed. High resolution of rGO image (B) has distinguished the graphene layers attributed to the multilayered graphene sheets. This atomic scale image of rGO shows the graphitic lattice fringes with a spacing of 0.34 nm. After simultaneous reduction of Pd salt and GO, the PdNPs are observed



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to uniformly distribute over the entire rGO sheet (image C and D). The presence of various nucleation sites on basal and edge plane of GO can promote the growth of PdNPs. The average particle size of PdNPs on rGO was estimated as 3.5 nm as shown in the histogram plot[inset: Figure 4(D)]. It has been seen that the shape of PdNPs arethree dimensional (image E). Various structures of PdNPs may be attributed to the presence of different phytochemicals during nucleation of Pd salt on GO sheets (image E).The atomic scale image of PdNPs has shown in image (F), where the lattice spacing has been estimated as 0.225 nm and 0.203 nm corresponding to (111) and (200) planes, respectively. In the XRD pattern of PdNPs, we have various planes such as (111), (200), (220), (311) and (222) at 2θ: 40.4°, 47.08°, 68.21°, 82.03° and 86.79° positions. Thus, dominant peaks observed due to (111) and (200) planes in XRD pattern indicating the growth of PdNPs along (111) and (200) directions on rGO sheets which is a strong agreement with HR-TEM studies. FE-SEM images of prepared Pd-rGO nanocompositehave been recorded (Figure 7) to investigate the morphological structures. Image (A) shows the rGOsheets having 10 micron size and are found to be agglomerated on silicon wafer due to π-π stacking. This rGO sheet is multi-layered and few wrinkles are clearly visible at rGO surface. In image B, the Pd-rGO sheets are agglomerated on ITO surface after electrophoretic deposition due to π-π stacking between individual rGO sheets. However, PdNPs are not clearly visible due to the small features of nanoparticles on rGO sheets. Higher resolution image of TEM analysis has resolved PdNPs on rGO sheets as observed in Figure 6 (C and D). Co-immobilization of LIP and GLDH on Pd-rGO sheets exhibits a porous feature due to strong covalent interactions (image C). Enhanced accumulation of these protein molecules on Pd-rGO surface may be due strong to electrostatic interaction between positively charged amino acid residues and negatively charged PdNPs. Energy dispersive spectroscopic analysis (EDS) analysis has confirmed the enzyme functionalization on Pd-rGO surface (image D and E). Inset (image d


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and e) shows the atomic ratios for various elements present in Pd-rGO with and without enzyme molecules. In image E, additional elements such as phosphorous (P) and sodium (Na) confirm presence of enzyme on Pd-rGO surface. Figure 7 (F) shows tapping mode AFM image of Pd-rGO sheets. In the height profile image of rGO [inset Figure 7 (F)], the average height of Pd-rGO is found to be 1.2 nm may be due to agglomeration. Electrochemical characterization studies Electrochemical characterizations of GO, rGO, Pd-rGO based electrodes and bioelectrode have been performed using CV. (Figure 8 a). There is a significant increase in the oxidation current (from 465.85µA to 628.10µA) after the green reduction of GO. This may be due to the fact that GO is typically insulating in nature, high sheet resistance and deficiency of percolating pathways among sp2 carbon network to permit classical carrier transport to take place.[5]In the case of Pd-rGO nanocomposite, the oxidation peak current has been further increased to 793.25 µA, which may be due to decreased electrostatic repulsion between negatively charged oxygenated groups and negatively charged ferro/ferricyanide redox probe after green reduction. Additional increase in the oxidation current for Pd-rGO nanocomposite may be attributed to the incorporation of in situ grown PdNPs which reduces tunnelling distance for electron conduction between electrode and electrolyte. After the coimmobilization of LIP-GLDH onto the Pd-rGO based transducer surface, the current has been found to decrease due to the insulating nature of enzyme molecules creating a hindrance in the electron transport between bulk solution and conducting current collector. Figure [S1] shows CV curves for the fabricated bioelectrode as a function of scan rate ranging from 10-100 mV/s. Magnitude of anodic and cathodic current has increased linearly and corresponding peaks are shifted towards the positive and negative potential, respectively, indicating uniform facile charge transfer [Eq. (2) and (3)]. The proportional increase in the



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anodic and cathodic peaks potential as a function of scan rate indicating the electrochemical reaction is a diffusion controlled process [Fig.S1, Inset (i)]. ./

[] = 5.6 × 10" [] + 71.04 × 10-4 [2%/'(] 1/2× [%)*+,-

0

./

) [] = −1.03 × 10-4 [] – 9.4 × 10-5 [2%/'(]1/2× [%)*+,-

0

]½ ;R2=0.99

]½;R2= 0.99

(2) (3)

The number of surface catalytic atoms present in the bioelectrode can be determined by calculating the charge associated in the process of adsorption/desorption. Smith et al. have demonstrated the electrical charge (Q) as an integral of cell current (I) with respect to time using the following relation; Q=5 6, 55.Further, total charge for the adsorbate ion at the electrode surface (Qm) and charge attached with monolayer coverage of the defined adsorbate (Qad) can be utilized for the determination of electrochemical surface area (Aecin cm-2) using the following Equation 4. 78 =

9:; 9
? BC DCE@

FG H

(5)

where R is the gas constant, T is the temperature, n is the number of electrons, F is Faraday constant, A is the effective area of the electrode and C is known as the concentration of redox couple in the bulk solution. The Ke value for biologically reduced GO has been found to be increased almost 2-folds (1.35× 10-4 cm/s) compared to that of GO (0.73× 10-4 cm/s) indicating decrease in the negatively charged oxygenated groups. While, further increase in the Ke value (1.47× 10-4 cm/s) for PdNPs embedded rGO sheet creating additional channels for electron conduction resulting in enhanced heterogeneous electron transfer. Triglyceride detection The electrochemical response studies of the fabricated bioelectrode have been investigated as a function of TB concentration for a wide detection range from 25 to 400 mgdL-1 in presence of PBS of pH 7.4containingferro/ferri cyanide as a redox probe using CV. As indicated in Fig. 8c, the CV peak current has been found to increase proportionally with respect to the concentration of TB. The biochemical reaction mechanism for the current generation has been explained in the Figure S2. LIP enzyme initially hydrolyse the TB resulting into the



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production of fatty acid and glycerol. In the second step, glycerol has been oxidized by GLDH enzyme in presence of NAD+ which acts as electron acceptor and produces NADH, dihydroxyacetone and hydrogen ions. Then, finally NADH re-oxidized into NAD+ by releasing an electron, which can be detected using the fabricated electrochemical electrode. The fabricated biosensor exhibits an excellent sensitivity of 5.1×10-3 mA mgdL-1, which may be attributed to the fast electron transfer between the enzymes and the electrode surface. Marccus et al. have described a model for distance dependent electron transfer between redox centre of the enzyme andGO.56 Since the redox centres of the enzymes lies deeply in the protein shell making electrochemical electron transfer very slow. In this regard, rGO layers known to decrease the distance between redox centre and transducer exchanging electrons through all positions increasing redox current. In addition, Pumera et al. have described the role of oxygen containing groups on the rGO surface towards the electrocatalysis of various enzymes influencing the property of heterogeneous charge transfer44.PdNPsavailable on the surface of rGO offers the additional surface area for the immobilization of the LIP-GLDH due to its strong electrostatic interaction with PdNPs. This results into the improved sensitivity and the detection limit as maximum number molecules of TB can react with immobilized LIP-GLDH resulting into more number of electrons. Additionally, it has been reported that PdNPs on the rGO surface offers additional channels for the direct electron transport may be the principle reason in achieving the superior sensitivity 57. The low detection limit of fabricated biosensor was found to be 25 mg/dL indicating that the fabricated biosensor have potential to detect the triglyceride even at very low concentrations. The proposed biosensor shows a prompt response time of 10 s (Figure S3). Further, the Michaelis–Menten constant (I. ) that determines the affinityof enzyme with the bioanalytewas calculated as 0.145 mgdL-1using Hanes-Woolf plot (Figure S4). The lower value of Km indicates the enhanced affinity of enzyme with the TB on the biosynthesized PdrGO transducer surface. This value is lowest among various other biosensors reported for TB


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detection as shown in Table 258-62. This may be due to better conformational orientation of the enzyme onto the biomaterial based transducer surface leading to improved interactions with TB. It is known the higher serum TB, a kind of triglyceride (TG) levels are directly associated with the risk of atherosclerosis and hyperlipidaemia and according to American heart association, the normal range in men is 40– 160 mgdL-1 and in women 35–135 mgdL-1.Apart from coronary diseases, higher level of TG is also responsible several other disorders including diabetes mellitus, nephrosis and liver obstruction. Pd-rGO composite synthesized using green synthesis has been proved to be an excellent matrix for the fabrication of ultrasensitive biosensor which can detect a wide range of TG concentration. To evaluate the specificity of the fabricated biosensor, the effect of possible interferents present in the blood such as cholesterol, glucose, urea, uric acid and ascorbic acid have been monitored using CV technique (Figure S5).The change in the oxidation current has been measured in PBS containing an equal amount (1: 1) of TB (200 mg dL-1) and interferent (Int) using Eq. 6. % Interference =

IAQ − RST (6) GU

Here, ITB is the current produced for detection of TB and IInt is the current observed in the presence of different interferents. The interference in the current has been found to be negligible with maximum % inference of 0.8 in case of uric acid. Figure S6 shows reproducibility test of the fabricated biosensor for eight different bioelectrodes with constant sensor surface. It has been found that all bioelectrodes shows negligible variation in the current response as evidenced by the relative standard deviation (RSD) of % (mean value = 801.5µA). The low RSD of this fabricated Pd-rGO based biosensor indicates good precision. Stability studies of the Pd-rGO biomaterial based biosensor have been performed by measuring the current response for 200 mg/dL concentration at the regular interval of 7 days



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(Figure S7). The bioelectrode exhibits 85% of the initial current response even after 6 weeks indicating bioelectrode can be utilized for at least 6 weeks when stored at 4ºC. CONCLUSION We have developed a facile, green and in situ route for simultaneous reduction of both graphene oxide and palladium salt in which the well dispersed and uniformly decorated PdNPs on rGO sheets. The natural spice (extract of FS) has acted as a reducing, stabilizing and decorating agent for the formation of Pd-rGO nanocomposite. This novel platform has been utilized for TB detection by co-immobilization of LIP-GLDH using covalent coupling chemistry. Due to excellent electrochemical properties the fabricated biosensor shows enhanced sensing characteristics such as sensitivity of 1.3 µA mgdL-1cm-1 in a wide detection range (25 - 400 mgdL-1). The value of Michaelis–Menten constant has been found as 0.14 mgdL-1with almost negligible interference from various possible interferents such as cholesterol, glucose, ascorbic acid, urea and uric acid. The biosensors shows improved biosensing characteristics compared to other reported in literature (Table 2). Further quick response, stability and repeatability were the additional features making this biologically reduced Pd-rGO nanocomposite as an effective transducer for the development of POC diagnostics devices. Supporting Information: A sheet containing supplementary information has been attached separately. ACKNOWLWDGMENTS We thank Director CSIR-NPL, New Delhi, India for the facilities. We are thankful to Dr. A.M.Biradar for his consistent support and encouragement. Authors are highly thankful to Prof. B.D. Malhotra from Delhi Technological University for his sincere guidance. C. S and Md.A.A. are thankful to CSIR, for the award of Senior Research Fellowships. Authors are thankful to Dr.Ved V. Agrawal, Dr.Bhanu Pratap Singh and Dr.Vidyanand Singh for AFM,


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Raman and HR-TEM studies, respectively. We acknowledge the financial support received from CSIR (ESC-0103).

.



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of oxygen moieties at graphene surface on gold nucleation and growth. Chem. Mater. 2009, 21, 4796-4802. (44) Pumera, M. Electrochemistry of graphene: New horizons for sensing and energy storage. Chem. Rec.2009, 9, 211-223. (45) Marcano, D. C.; Kosynkin, D. V.; Berlin, J. M.; Sinitskii, A.; Sun, Z.; Slesarev, A.; Alemany, L. B.; Lu, W.; Tour, J. M. Improved synthesis of graphene oxide. ACS Nano.2010, 4, 4806-4814. (46) Wu, Z. S.; Pei, S.; Ren, W.; Tang, D.; Gao, L.; Liu, B.; Li, F.; Liu, C.; Cheng, H. M. Field emission of single layer graphene films prepared by electrophoretic deposition. Adv. Mater. 2009, 21, 1756-1760. (47) Kudin, K. N.; Ozbas, B.; Schniepp, H. C.; Prud'Homme, R. K.; Aksay, I. A.; Car, R. Raman spectra of graphite oxide and functionalized graphene sheets. Nano Lett .2008, 8, 3641. (48) Ferrari, A. C.; Robertson, J. Interpretation of Raman spectra of disordered and amorphous carbon. Phys. Rev. B.2000, 61, 14095-14107. (49)Ferrari, A. C. Raman spectroscopy of graphene and graphite: disorder, electron-phonon coupling, doping and nonadiabatic effects. Solid State Commun. 2007, 143, 47-57. (50)Cancado, L. G.; Takai, K.; Enoki, T.; Endo, M.; Kim, Y. A.; Mizusaki, H.; Jorio, A.; Coelho, L. N.; Magalhaes-Paniago, R.; Pimenta, M. A. General equation for the determination of the crystallite size L a of nanographite by Raman spectroscopy. Appl. Phys. Lett .2006, 88, 163106-08. (51)Li, D.; Miller, M. B.; Gilje, S.; Kaner, R. B.; Wallace, G. G. Processable aqueous dispersions of graphene nanosheets. Nat. Nanotechnol.2008, 3, 101-105. (52) Paredes, J. I.; Villar-Rodil, S.; Solis-Fernandez, P.; Martinez-Alonso, A.; Tascon, J. M. D. Atomic force and scanning tunneling microscopy imaging of graphene nanosheets derived from graphite oxide. Langmuir.2009, 25, 5957-5968.


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(53) Yang, X.; Li, Q.; Wang, H.; Huang, J.; Lin, L.; Wang, W.; Sun, D.; Su, Y.; Opiyo, J. B.; Hong, L. Green synthesis of palladium nanoparticles using broth of Cinnamomum camphora leaf. J. Nanopart. Res.2010, 12, 1589-1598. (54) Yonezawa, T.; Imamura, K.; Kimizuka, N. Direct preparation and size control of palladium nanoparticle hydrosols by water-soluble isocyanide ligands. Langmuir.2001, 17, 4701-4703. (55) Watt-Smith, M. J.; Friedrich, J. M.; Rigby, S. P.; Ralph, T. R.; Walsh, F. C. Determination of the electrochemically active surface area of Pt/C PEM fuel cell electrodes using different adsorbates. J. Phys. D: Appl. Phys. 2008, 41, 174004-012. (56) Martins, M. V. A.; Pereira, A. R.; Luz, R. A. S.; Iost, R. M.; Crespilho, F. N. Evidence of short-range electron transfer of a redox enzyme on graphene oxide electrodes. Phys. Chem. Chem. Phys.2014, 16, 17426-17436. (57)Xiang, J.; Drzal, L. T. Electron and phonon transport in Au nanoparticle decorated graphene nanoplatelet nanostructured paper. ACS Appl. Mater. Interfaces.2011. 3, 13251332. (58)Pundir, C. S. Construction of an amperometric enzymic sensor for triglyceride determination. Sens. Actuators, B. 2008, 133, 251-255. (59)Pundir, C. S.; Sandeep Singh, B.; Narang, J. Construction of an amperometric triglyceride biosensor using PVA membrane bound enzymes. Clin. Biochem. 2010, 43, 467472. (60) Dhand, C.; Solanki, P. R.; Sood, K. N.; Datta, M.; Malhotra, B. D. Polyaniline nanotubes for impedimetric triglyceride detection. Electrochem. Commun. 2009, 11, 14821486. (61) Liao, W.-Y.; Liu, C.-C.; Chou, T.-C. Detection of triglyceride using an iridium nanoparticle catalyst based amperometric biosensor. Analyst.2008, 133, 1757-1763.



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(62) Narang, J.; Pundir, C. S. Construction of a triglyceride amperometric biosensor based on chitosan-ZnO nanocomposite film.Int. J. Biol. Macromol.2011, 49, 707-715.

Figure captions: Figure1:Composition of Fenugreek seeds with respect top hytochemicals. Figure 2: B, H and T sites in hexagonal graphene sheet and DFT predicted position for PdNPs. Figure 3: Schematic representation on the fabrication of Pd-rGO nanocomposite and its utilization for triglyceride detection. Figure 4: Raman spectra for GO and biologically reduced GO (4a), UV visible spectrum of GO, biologically reduced GO, GO-PdCl2 solution before reaction, Pd-rGO solution and for FS extract solution (4b). Figure 5: FT-IR spectra of GO, rGO, Pd-rGO and FS extract. Figure 6: HR-TEM image of multi-layered rGO sheet and SAED pattern of biologically reduced GO (Inset) (a), atomic scale image of rGO shows the graphitic lattice fringes (b), uniformly decorated PdNPs onto the rGO sheet (c, d) and particle size distribution curve for PdNPs onto the rGO (inset d), multi-shaped Individual PdNPs (e), atomic scale image of PdNP with lattice spacing (f). Figure 7: SEM images of agglomerated rGO sheets (a), Pd-rGO deposited onto ITO using EPD technique (b), Immobilization of LIP-GLDH onto Pd-rGO electrode (c),EDX analysis of Pd-rGO electrode (d), EDX analysis of enzyme immobilized Pd-rGO electrode (e) AFM analysis of Pd-rGO with height profile (Inset).


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Figure 8: CV characterization studies of GO, rGO, Pd-rGO and LIP-GLDH/Pd-rGO electrodes (a), EIS characterization studies of GO, rGO, Pd-rGO and LIP-GLDH/Pd-rGO electrodes (b), CV response of LIP-GLDH/Pd-rGO electrodes with different concentration of TB (c), Calibration curve between concentration and current for various concentrations from 25-400 mg/dl. FigureS1: CV analysis of LIP-GLDH/Pd-rGO electrode as a function of scan rate [10-100 mV/s] and anodic/cathode peak current with respect to square root of scan rate (Inset). Figure S2: Electrochemical reactions pathway and generation of electron due to the reaction. FigureS3:Hanes-Woolf plot for the biosensing response of LIP-GLDH/Pd-rGO. Figure S4: Chronoamperometric responses of LIP-GLDH/Pd-rGO. Figure S5: Interference study of LIP-GLDH/Pd-rGO electrode in presence of various possible interferents. Figure S6: Repeatability studies of LIP-GLDH/Pd-rGO electrode. Figure S7: Stability studies of LIP-GLDH/Pd-rGO electrode. Table1: Equivalent circuit elements of different electrodes. Table 2: Comparison of various biosensing parameters of fabricated biosensor with other triglyceride biosensors reported in literature.



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Figure and Table

Figure 1


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Figure 2

Figure 3



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Figure 4

Figure 5


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Figure 6

Figure 7



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Figure 8


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Table1: Equivalent circuit elements of different electrodes

Electrode

Rs(kΩ)

RCT (kΩ)

Cdl(F)

GO

1.46× 101

2.77

6.35× 10-5

rGO

2.87× 101

1.67

1.04× 10-4

Pd-rGO

3.05× 101

1.60

9.05× 10-5

LIP-GLDH/Pd-rGO

2.05× 101

2.27

5.81× 10-5

Table 2: Comparison of various biosensing parameters of fabricated biosensor with other triglyceride biosensors reported in literature

Working electrodes

Linearity range (mg/dL)

Sensitivity

8.8-309.7

Lower detection limit (mg/dL) 17.69

Cellulose acetate membrane PVA membrane

Stability

Reference

…….

Km value (mg/d L) 775.2

…….

18.58

……..

294.6

….

84×10-6 µA (mg/dL)-

…..

50% loss in 50 days …..

Minakshi et al.58 Pundir et al.59

49.55–199.11

Iridium nano-particle modified carbon paste

0 –884.95

Polyaniline nanotubes based film

25–300

25

2.59 Ω-1(mg/dL)-1

46.69

……

Dhand et al.60

Chitosan–ZnO nanocomposite film Pd-rGO nanocomposite

50–650

20

0.89 mg/dL-1 µA-1

……

Narang et al.62

25

1.3 µA mgdL-1cm-1

0.14 mg/dL

50% loss in 7 months 15% loss in 6 weeks

25-400

Liao et al.61

1


Current work


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For table of content use only

Green Synthesis of Graphene Based Biomaterial using Fenugreek Seeds for Lipid Detection

Chandan Singh, Md. Azahar Ali, GajjalaSumana

Demonstrating the use of a natural spice, fenugreek seeds for the development of graphene based biomaterial for the efficient detection of lipid.


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Green Synthesis of Graphene Based Biomaterial Using Fenugreek

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