Etlingera Elatior-Mediated Synthesis of Gold Nanoparticles and Their Application As Electrochemical Current Enhancer

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Article Etlingera elatior-Mediated Synthesis of Gold Nanoparticles and Their Application as Electrochemical Current Enhancer

Farah Asilah Azri 1,* , Jinap Selamat 1,2,* , Rashidah Sukor 1,2 , Nor Azah Yusof 3, Nurul Hanun Ahmad Raston 4, Noordiana Nordin 1 and Nuzul Noorahya Jambari 1,2 1 Laboratory of Food Safety and Food Integrity, Institute of Tropical Agriculture and Food Security, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 2 Department of Food Science, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 3 Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia 4 School of Bioscience and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia * Correspondence: [email protected] (F.A.A.); [email protected] (J.S.)

Academic Editor: Barbara Bonelli Received: 8 May 2019; Accepted: 15 July 2019; Published: 29 August 2019

Abstract: This work presents a simple green synthesis of gold nanoparticles (AuNPs) by using an aqueous extract of Etlingera elatior (torch ginger). The metabolites present in E. elatior, including sugars, proteins, polyphenols, and ﬂavonoids, were known to play important roles in reducing metal ions and supporting the subsequent stability of nanoparticles. The present work aimed to investigate the ability of the E. elatior extract to synthesise AuNPs via the reduction of gold (III) chloride hydrate and characterise the properties of the nanoparticles produced. The antioxidant properties of the E. elatior extract were evaluated by analysing the total phenolic and total ﬂavonoid contents. To ascertain the formation of AuNPs, the synthesised particles were characterised using the ultraviolet-visible (UV-Vis) spectroscopy, Fourier transform infrared (FTIR) spectroscopy, high-resolution transmission electron microscopy (HRTEM), energy-dispersive X-ray (EDX) microscopy, and dynamic light scattering (DLS) measurement. The properties of the green synthesised AuNPs were shown to be comparable to the AuNPs produced using a conventional reducing agent, sodium citrate. The UV-Vis measured the surface plasmon resonance of the AuNPs, and a band centered at 529 nm was obtained. The FTIR results proved that the extract contained the O-H functional group that is responsible for capping the nanoparticles. The HRTEM images showed that the green synthesized AuNPs were of various shapes and the average of the nanoparticles’ hydrodynamic diameter was 31.5 0.5 nm. Meanwhile, ± the zeta potential of 32.0 0.4 mV indicates the high stability and negative charge of the AuNPs. − ± We further successfully demonstrated that using the green synthesised AuNPs as the nanocomposite to modify the working surface of screen-printed carbon electrode (SPCE/Cs/AuNPs) enhanced the rate of electron transfer and provided a sensitive platform for the detection of Cu(II) ions.

Keywords: gold nanoparticles; green synthesis; Etlingera elatior; torch ginger; electrochemical

1. Introduction The application of metallic nanoparticles is gaining traction in various ﬁelds due to their unique electrochemical and optical properties. These unique properties make them an ideal nanomaterial to be used in biosensor applications. The electrochemical properties of AuNPs can enhance the electrode conductivity by facilitating electron transfer, and thus improving the sensitivity. Metallic nanoparticles of explicit sizes and morphologies can be readily synthesised by means of chemical and physical

Molecules 2019, 24, 3141; doi:10.3390/molecules24173141 www.mdpi.com/journal/molecules Molecules 2019, 24, 3141 2 of 15 methods [1]. The AuNPs provide advantages in many applications including biomedical, drug delivery, photothermal therapy, tissue or tumour imaging, labelling and sensing [2,3]. Increasing awareness towards green chemistry and other biological processes in recent years has led the need to develop an eco-friendly approach for the synthesis of AuNPs. This is largely due to the relative ease of the production, good control of sizes and shapes, unique optical and electronic properties, and good biocompatibility of the AuNPs. Green synthesis or plant-mediated biological synthesis of nanoparticles is drawing attention from the scientific community due to its cost-effectiveness, simplicity, ease of up-scaling, and safety. Green synthesis is a bottom-up approach where the nanoparticles are built from smaller entities and then assembled to produce the final particles [4]. The AuNPs synthesis requires a protective or stabilising agent to adsorb onto the surface of the newly formed nanoparticles to prevent particles agglomeration and further growth. The most commonly used stabilising agents reported are sodium citrate (Na3C6H5O7), transferrin, sodium borohydride (NaBH4), and cetyltrimethylammonium bromide (C19H42BrN), which are known as the chemical method that can subsequently produce toxic wastes [5]. Therefore, alternative non-toxic reagents are often sought after to improve the biocompatibility of AuNPs. Several biological agents such as bacteria, fungi, algae and plants have been applied in the green synthesis of metallic nanoparticles. However, the use of plant materials for the synthesis of AuNPs has been demonstrated to be more advantageous than microbiological-based methods as (1) it eliminates the elaborate process of maintaining the bacterial and fungal cultures [6], (2) the nanoparticles synthesised by plant extract are shown previously to be more stable, and (3) the production rate is faster than those synthesised using microorganisms [7]. Plant extracts act as reducing agents as well as stabilising agents, which are believed to greatly influence the characteristics of the nanoparticles [8]. The reduction process of metal ions during the formation of nanoparticles is affected by several factors, such as the nature of plant extracts that contains active biomolecules at different combinations and concentrations, pH of the reaction mixture, incubation temperature, reaction time, and electrochemical potential of the metal ions [9]. Plant biomolecules, which are a large group of polyphenolic compounds that comprise of anthocyanins, isoflavonoids, flavonols, chalcones, falvones, and flavanones, have been shown to actively chelate and reduce metal ions into nanoparticles [10,11]. The protective and reductive activities of this plant biomolecules are accountable for the reduction of gold ions. There have been a number of reports on the synthesis of various shapes of AuNPs using extracts from fruits, stalks, or leaves of different plants such as Ocimum sanctum (holy basil) [12], Nepenthes khasiana (pitcher plant) [13], Azadirachta indica (neem) [14], Basella alba (vine spinach) [15], Allium sativum (garlic) [16], Ficus benghalensis (banyan) [17], and Carica papaya (papaya) [18] but with different efficacies. Etlingera elatior or commonly known as torch ginger is a fast-growing plant that is widely cultivated and abundantly available throughout Southeast Asia. The inflorescence E. elatior is a fleshy pink flower that grows at the tip of stalks with bracts spreading. The E. elatior flowers have immense ornamental values and these young flowering shoots are commonly added to various dishes especially in Malaysia, Indonesia, and Singapore to improve the taste and smell. Previous studies on the characterisation of the chemical composition of E. elatior extracts by GC-MS revealed more than 30 major organic compounds, which includes fatty alcohols (Z-11-Pentadecenol, dodecanol, 1-Hexadecanol), fatty acids (dodecanoic acid, tetradecanoic acid), fatty aldehydes (dodecanal, 1-Tetradecyl acetate), tannins, flavonoids, saponins, and steroids [19–21]. In addition to the antioxidative, anticancer, and antimicrobial properties of these organic compounds [19,20,22], the abundance of these compounds in E. elatior makes the plant a promising source of reducing and stabilising agents for the green synthesis of AuNPs. In this study, we aimed to support and contribute additional evidence on the ability of E. elatior as a reductant and stabiliser in the AuNP synthesis [12], and also explore the potential application of the E.elatior-mediated synthesised AuNPs as an electrode modifier. The resulting modified electrode was further applied as an electrochemical metal ion detection system to measure copper ions by differential pulse stripping voltammetry in the aqueous solution. Molecules 2019, 24, 3141 3 of 15

2. Results and Discussion

2.1. Determination of Phenolic and Flavonoids Compounds Different plant sources contain different concentrations and combinations of reducing agents that may affect the characteristics of the nanoparticles [8]. The reduction of the metal ions was attributed to the phenolics, terpenoids, polysaccharides and flavones compounds present in the extract [9]. Total phenolic compounds (TPC) in plants are known to act as free radical scavengers and it is believed that the antioxidant activity of the most plant produce is mostly due to the presence of phenolic compounds [13]. The antioxidative mechanism of polyphenolic compounds depends on their hydrogen donating and metal ion chelating capabilities [14,15]. Gallic acid (GA) was used as the standard for the calibration curve and TPC was expressed as the gallic acid equivalent (GAE) in mg per 100 g of the sample. The calibration equation for gallic acid was;

y = 0.0112x + 0.0253 (R2 = 0.999) (1) where y is the absorbance and x is the GA concentration in mg/mL. In this study, the TPC concentration of the E. elatior extract was 193.5 1.03 mg GAE/100 g sample. ± We then determined the ﬂavonoids content of the E. elatior extract based on the aluminium chloride colourimetric assay. The calibration equation for quercetin was;

y = 0.0024x + 0.0113 (R2 = 0.996) (2) where y is the absorbance and x is the QE concentration in mg/mL. The total flavonoids content (TFC) of the E. elatior extract was 135.5 1.34 mg QE/100 g sample. Flavonoids are the most common and ± extensively distributed single group of phenols found in plants [16]. Flavanoids, as highly effective antioxidants, can chelate metal ions with their carbonyl groups or π-electrons and avert metal-initiated lipid oxidation by forming complexes with metal ions [15]. These compounds are believed to be involved during the initiation of nanoparticles nucleation and further aggregation or bioreduction stage [10]. The tautomeric conversions of flavonoids from the enol-form to the keto-form may release a volatile hydrogen atom that can reduce metal ions to form nanoparticles [10]. The type of extraction solvent used has been shown to greatly influence the amount of antioxidants extracted from the E. elatior flower [17]. Sepahpour et al. [17] and Wijekoon et al. [18] reported that water was the least effective solvent for the phenolic extraction from E. elatior compared to other organic solvents used, such as methanol, ethanol and acetone. Table1 shows the amount of TPC and TFC extracted using different percentages of methanol. Based on the results, 50% of methanol in the extraction solvent produced higher TPC while 100% of methanol produced more the TFC content. However, in this study, we used 10% of methanol as the extraction solvent to minimise any detrimental effect during the AuNPs synthesis procedure. We discovered that the amount of phenolics and flavonoids extracted from the fresh E. elatior using water with 10% of methanol was sufficient to be used as the reducing and stabilising agent for the AuNPs synthesis.

Table 1. Total phenolic and total ﬂavonoid content in the E. elatior extract extracted using diﬀerent percentages of methanol in the extraction solvent.

Extraction solvent TPC (mg GAE/100 g) TFC (mg QE/100 g) References 100% water 90.7 1.7 182.5 3.2 [18] ± ± 100% methanol 361.2 17.1 762.8 44.5 [18] ± ± 90% methanol 431.4 25.8 632.6 51.2 [18] ± ± 50% methanol 615 14.6 717.6 41.0 [18] ± ± 10% methanol 193.5 1.0 135.5 1.3 This study ± ± Molecules 2019, 24, 3141 4 of 15

2.2. Characterisation of Synthesised Gold Nanoparticles

2.2.1. Characterisation via Physical Observation and Ultraviolet-Visible Spectroscopy Molecules 2019, 24, x FOR PEER REVIEW 4 of 15 A rapid colour change of gold solution from light yellow to deep red in the reaction mixture was observedA rapid colour shortly change after of the gold addition solution of from the E. light elatior yellowextract. to deep The red complete in the reaction reduction mixture of gold was ions wasobserved observed shortly within after 15the min. addition The colourof the E. exhibited elatior extract. by metallic The complete nanoparticles reduction was of due gold to theions coherent was excitationobserved within of all free 15 electronsmin. The withincolour theexhibited conduction by metallic band, leadingnanoparticles to an in-phase was due oscillation to the coherent known as excitation of all free electrons within the conduction band, leading to an in-phase oscillation known localised surface plasmon resonance (LSPR) with a specific wavelength of incident light [19]. The LSPR as localised surface plasmon resonance (LSPR) with a specific wavelength of incident light [19]. The of AuNPs produced a strong absorbance band in the visible region that could be measured by the LSPR of AuNPs produced a strong absorbance band in the visible region that could be measured by UV-Vis spectroscopy. The colour change indicates the different sizes and shapes of AuNPs due to the the UV-Vis spectroscopy. The colour change indicates the different sizes and shapes of AuNPs due excitation of surface plasmon vibrations. Figure1a shows the di fferences in the colour of the AuNPs to the excitation of surface plasmon vibrations. Figure 1a shows the differences in the colour of the solutions. The colour intensity of the AuNPs solution synthesised using the E. elatior extract was higher AuNPs solutions. The colour intensity of the AuNPs solution synthesised using the E. elatior extract thanwas higher to the AuNPsthan to the solution AuNPs synthesised solution sy vianthesised the citrate via the reduction citrate reduction method. method. Our initial attempt attempt to to synthesise synthesise AuNPs AuNPs using using a mildly a mildly acidic acidic pristine pristine extract extract of E. of elatiorE. elatior resultedresulted inin the formation formation of of a adark dark purple purple solution. solution. Furthe Furtherr LSPR LSPR confirmation confirmation of the of synthesised the synthesised AuNPs AuNPs by bythe theUV-vis UV-vis spectroscopic spectroscopic analysis analysis showed showed that th thate SPR the peak SPR for peak the green for the synthesised green synthesised AuNPs using AuNPs usingthe pristine the pristine E. elatiorE. elatiorextractextract was observed was observed at 574 at nm 574 (Figure nm (Figure 1b).1 b).Here Here we weassumed assumed that that the the nanoparticlesnanoparticles produced produced were were in in a large a large size size and and tend tend to aggregate. to aggregate. This Thisis because is because the aggregation the aggregation of ofAuNPs AuNPs subsequently subsequently resulted resulted in the in theshift shift of the of theabsorption absorption band band to longer to longer wavelength wavelength due dueto the to the electricelectric dipole-dipole interaction interaction and and coupling coupling between between the the plasmons plasmons of neighbouring of neighbouring particles particles in the in the formform of aggregates [20]. [20].

1.6 (a) (b) 529 1.4 Citrate Reduction Green Synthesis (pH 7.5) 1.2 Green Synthesis (pH 4.2) 1.0 520

0.8 574

Absorbance 0.6

0.4

0.2

0.0 400 450 500 550 600 650 700 750 800 (i) (ii) (iii) Wavelength (nm)

Figure 1. (a) Gold nanoparticles (AuNPs) solutions; i. red wine colour of AuNPs solution synthesised byFigure the 1. citrate (a) Gold reduction nanoparticles method, (AuNPs) ii. deep solutions; red colour i. red ofwine AuNPs colour solution of AuNPs synthesised solution synthesised by the green synthesisby the citrate method reduction (pH 7.5), method, and iii. ii. purple deep colourred colo ofur AuNPs of AuNPs solution solution synthesised synthesised by the by green the green synthesis methodsynthesis (pH method 4.2). (b(pH) UV-visible 7.5), and spectrumiii. purple of colour synthesised of AuNPs AuNPs solution by respective synthesised methods by withthe green the scale ofsynthesis wavelength method between (pH 4.2). 400 ( andb) UV-visible 800 nm. spectrum of synthesised AuNPs by respective methods with the scale of wavelength between 400 and 800 nm. However, by adjusting the pH of the E. elatior extract from 4.2 to 7.5, a deep red solution was producedHowever, and by a shiftadjusting in the the SPR pH wavelengthof the E. elatior to extract 529 nm from was 4.2 observed. to 7.5, a deep pH red was solution shown was to play aproduced signiﬁcant and role a shift in controllingin the SPR thewavelength size and to morphology 529 nm was ofobserved. the produced pH was nanoparticles shown to play [21 a, 22]. Singhsignificant and Srivastavarole in controlling[23] reported the size that and there morphology was a gradual of the produced shift in the nanoparticles absorption [21,22]. on the synthesisSingh ofand AuNPs Srivastava using [23] the reported black cardamom that there extract was a whengradual pH shift was in increased the absorption from highly on the acidic synthesis to neutral. of ThisAuNPs wavelength using the black shift indicatescardamom that extract the particlewhen pH size was was increased reduced, from and highly vice acidic versa to when neutral. the pHThis was increasedwavelength from shift neutral indicates to highly that the alkaline. particle There size was was a slightreduced, red and shift vice in the versa SPR when wavelength the pH at was 529 nm forincreased the E. elatiorfrom -mediatedneutral to highly AuNPs alkaline. compared There to the was peak a slight of citrate-capped red shift in the AuNPs SPR wavelength at 520 nm. at However, 529 nm for the E. elatior-mediated AuNPs compared to the peak of citrate-capped AuNPs at 520 nm. However, the sharp and symmetrical SPR peaks were indicative of small spherical nanoparticles. Based on the absorbance profile, the higher absorbance peak of the green synthesised AuNPs

Molecules 2019, 24, 3141 5 of 15 the sharp and symmetrical SPR peaks were indicative of small spherical nanoparticles. Based on the absorbance proﬁle, the higher absorbance peak of the green synthesised AuNPs compared to the citrate-capped AuNPs suggests that a higher concentration of the AuNPs were produced.

2.2.2. Characterisation via Fourier Transform Infrared Spectroscopy The functional groups of the compounds responsible for the formation and stabilization of AuNPs were identified using FTIR spectroscopy. The AuNPs that were synthesised using plant extracts were shown to be coated with a thin layer of organic material that acted as capping agents [24]. Green synthesis causes the nanoparticles to be surrounded by the plant metabolites including sugars, terpenoids, polyphenols, alkaloids, phenolic acids and proteins. These plant metabolites play important roles in the reduction of metal ions into nanoparticles and supporting their subsequent stability [10]. The FTIR main signals are indexed in Figure2 while the details on the IR bands were provided in the supplementary materials (Table S1). The FTIR spectrum of the E. elatior extract in Figure2a shows the 1 1 band at 3254 and 1635 cm− . The intense broad absorbance at 3254 cm− is the characteristic of the hydroxyl (stretch O-H bonded, strong broad) functional group in alcohols and phenolic compounds. 1 The highest absorption peak at 3254 cm− reflected the -OH group that were potentially responsible 1 for the reducing properties of the E. elatior extract [25]. Meanwhile, the band at 1635 cm− could be amide I band of the proteins released by the E. elatior or C=C groups/aromatic rings [26]. All these peaks reveal the presence of highly concentrated alcohol or phenol, aromatic amines and amide (I) of proteins in the extract. The FTIR spectrum of the AuNPs synthesised by the green synthesis method (Figure2b) shows 1 bands at 1368, 3159, and 3380 cm− along with other small bands. The decrease in the peak intensity observed after the encapsulation of chloroaurate ion suggest the possible contribution of the mentioned 1 groups. The band at 1368 cm− that corresponds to the C-N stretching vibration of aliphatic amines or alcohols/phenols may be responsible for the reduction and capping of AuNPs. Meanwhile, the band at 1 3380 and 3159 cm− correspond to the amide II bands (N-H) of proteins. The N-H vibrational bands become weaker and broader in the spectrum of stabilised AuNPs. This indicates that the AuNPs synthesised using the E. elatior extract were surrounded by some proteins and metabolites. Figure2c represents the FTIR spectrum of AuNPs synthesised by the citrate reduction method, which shows the 1 1 band at 3241, 1694, and 1538 cm− along with other small bands. The band at 3241 cm− represents strong and broad O-H stretching which refer to the intermolecular bonds of the molecules in sodium 1 citrate (Na3C6H5O7) that has three -OH groups in the structure. A peak present at 1694 cm− denotes the C=N stretching of imine/oxime compounds. Oximes are usually generated by the reaction of 1 hydroxylamine with aldehydes or ketones. The peak at 1538 cm− is due to strong N-O stretching from the nitro compound that was also present in sodium citrate. All three FTIR spectra (Figure2a–c) clearly show the changes in the -COOH group for -OH, i.e., 1 the hydroxyl group at the peak of 3254 cm− which appeared in the plant extract. However, the peak 1 was narrower and shifted to 3159 and 3241 cm− following the encapsulation of nanoparticles. These FTIR results support the finding in Section 2.1, which suggested that the E. elatior extract is rich in phenolics with sufficient amount hydroxyl and carboxyl groups. These hydroxyl and carboxyl groups possess oxidation-reduction abilities to bind metals and inactive them from chelation. Therefore, it can be suggested that the hydroxyl groups are the main contributor in the reduction of Au (III) to Au (0) through the oxidation of hydroxyl to the carbonyl group, which can be presented as:

0 + AuCl − + 3R-OH Au + 3R-O + 3H + 4Cl− (3) 4 → Molecules 2019,, 24,, 3141x FOR PEER REVIEW 66 of of 15

100 (a) 90

80 (b)

70 3159.17 3380.17 1368.43 (c) 60

50 1538.59 1694.20 % Transmittance % 1635.30 40

30 3241.00

20 3254.48

0 4000 3500 3000 2500 2000 1500 1000 500 Wavenumber (cm-1)

Figure 2. FTIR spectrum of (a) aqueous extract of E. elatior;(b) purified AuNPs synthesised by the Figure 2. FTIR spectrum of (a) aqueous extract of E. elatior; (b) purified AuNPs synthesised by the green synthesis method using the E. elatior extract, and (c) purified AuNPs synthesised by the citrate green synthesis method using the E. elatior extract, and (c) purified AuNPs synthesised by the citrate reduction method. reduction method. 2.2.3. Characterisation via High-Resolution Transmission Electron Microscopy and Energy-Dispersive X-ray2.2.3. Characterisation via High-Resolution Transmission Electron Microscopy and Energy- Dispersive X-ray The HRTEM images in Figure3 display clear shapes of the AuNPs which confirm their formation. The newlyThe HRTEM formed images AuNPs usingin Figure the E.3 elatiordisplayextract clear observedshapes of were the polydisperseAuNPs which and confirm consisted their of aformation. mixture ofThe spherical, newly formed triangular AuNPs and using rod-shaped the E. withelatior irregular extract observed contour particleswere polydisperse (Figure3a,c,e). and Theconsisted triangular of a andmixture rod-shaped of spheri particlescal, triangular formed (Figureand rod-shaped S3) were shownwith irregular to have highcontour surface particles areas while(Figures various 3a, 3c sizesand 3e). of sphericalThe triangular nanoparticles and rod-shaped were observed. particles Theformed nanoparticles (Figure S3) formed were shown were into thehave range high of surface 3–25 nm areas in size, while as thevarious diameter sizes measured of spherical manually. nanoparticles The morphologies were observed. of the green The synthesisednanoparticles AuNPs formed were were then in comparedthe range toof the3–25 AuNPs nm in synthesisedsize, as the usingdiameter the standardmeasured citrate manually. reduction The methodmorphologies (Figure of3 theb,d,f). green The synthesised citrate-capped AuNPs AuNPs were wasthen showncompared to be to morethe AuNPs uniformly synthesised spherical using in shapethe standard (Figure citrate3d) and reduction present inmethod almost (Figures similar size.3b, 3d Both and green 3f). The and citrate-capped citrate synthesised AuNPs AuNPs was shown were shownto be more to be uniformly stable with spherical no further in shape aggregation (Figure and 3d) colour and present changes in whenalmost the similar solutions size. were Both storedgreen forand up citrate to three synthesised months at AuNPs 4 ◦C. The were possible shown nucleation to be stable and with growth no forfurther different aggregation shapes ofand AuNPs colour is influencedchanges when by di thefferent solutions components were stored present for in up the to plantthree extract.months Based at 4 °C. on The the studypossible by nucleation Jiang et al. and [27] reducinggrowth for sugars different might shapes be responsible of AuNPs for is the influenc formationed by of different spherical components particles which present also preventsin the plant the nanoparticlesextract. Based fromon the aggregation. study by Jiang Meanwhile, et al. [27] flavones reducing correspond sugars might to the be fastresponsible nucleation for andthe formation growth of theof spherical flower-like particles structures which with also the prevents help of the steric nanoparticles hindrance offrom ketones aggregation. [27]. Well-defined Meanwhile, triangular flavones andcorrespond hexagonal to the shaped fast AuNPsnucleation were and believed growth to of be th synthesisede flower-like by structures the polyphenol with the molecules help of ofsteric the planthindrance extract. of ketones The reacted [27]. adjacentWell-defined phenolic triangular hydroxyls and ofhexagonal polyphenols shaped are AuNPs inductively were oxidized believed to to the be correspondingsynthesised by carbonyls the polyphenol and ketones molecules due to theof oxidation-reductionthe plant extract. The potential reacted of Au(III).adjacent phenolic hydroxyls of polyphenols are inductively oxidized to the corresponding carbonyls and ketones due to the oxidation-reduction potential of Au(III).

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(a) (b)

50 nm 50 nm

20 nm 20 nm

(e) (f)

10 nm 10 nm

FigureFigure 3. High-Resolution TransmissionTransmission Electron Electron Microscopy Microscopy (HRTEM) (HRTEM) images images of the ofE. the elatior-mediated E. elatior- mediatedAuNPs atAuNPs (a) 50,000X at (a) 50,000X magnification; magnification; (c) 100,000X (c) 100,000X magnification; magnification; (e) 250,000X (e) 250,000X magnification magnification and in andcomparison, in comparison, with AuNPs with AuNPs synthesised synthesised by sodium by sodium citrate atcitrate (b) 50,000X at (b) magnification;50,000X magnification; (d) 100,000X (d) 100,000Xmagnification; magnification; (f) 250,000X (f) 250,000X magnification. magnification.

The EDX spectrum proﬁles in Figure4 gives a clear indication that other elements were involved The EDX spectrum profiles in Figure 4 gives a clear indication that other elements were involved in the synthesis of the AuNPs. This analysis conﬁrmed that the gold was the highest elementary in the synthesis of the AuNPs. This analysis confirmed that the gold was the highest elementary composition. The detection of strong gold (Au) signal along with weak signals of carbon (C), nitrogen composition. The detection of strong gold (Au) signal along with weak signals of carbon (C), nitrogen (N) and oxygen (O) suggests the presence of biomolecules bound to the surface of the AuNPs from (N) and oxygen (O) suggests the presence of biomolecules bound to the surface of the AuNPs from the capping with the organic materials of the E. elatior extract (Figure4a). This infers the presence of the capping with the organic materials of the E. elatior extract (Figure 4a). This infers the presence of stabilising molecules on the green synthesised AuNPs. The EDX spectrum of AuNPs synthesised via stabilising molecules on the green synthesised AuNPs. The EDX spectrum of AuNPs synthesised via the citrate reduction method (Figure4b) shows that there is aluminium (Al) present as impurities in the the citrate reduction method (Figure 4b) shows that there is aluminium (Al) present as impurities in sample coming from the sample substrate but no N was present. Based on the EDX spectrum analysis, the sample coming from the sample substrate but no N was present. Based on the EDX spectrum the proposed green synthesis method has successfully produced 80.6% of gold as compared to 86.2% analysis, the proposed green synthesis method has successfully produced 80.6% of gold as compared by the citrate reduction method, which demonstrates its great potential as an alternative method to to 86.2% by the citrate reduction method, which demonstrates its great potential as an alternative synthesis AuNPs. method to synthesis AuNPs.

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(a) (b)

Figure 4. Energy Dispersive X-ray (EDX) spectrum showing the presence of gold elements and other Figure 4. Energy Dispersive X-ray (EDX) spectrum showing the presence of gold elements and other bioorganic components in (a) AuNPs synthesised by E. elatior;(b) AuNPs synthesised by sodium citrate. bioorganic components in (a) AuNPs synthesised by E. elatior; (b) AuNPs synthesised by sodium Insert graphs represent the elements distribution percentage in the samples. citrate. Insert graphs represent the elements distribution percentage in the samples. 2.2.4. Characterisation via Dynamic Light Scattering (DLS) 2.2.4. Characterisation via Dynamic Light Scattering (DLS) The DLS measurement is known to measure the shell thickness of a capping or stabilizing agent envelopingThe DLS the measurement synthesised is nanoparticles known to measure along the with she thell thickness actual size of a of capping the metallic or stabilizing core [20 ].agent DLS envelopingreveals the the hydrodynamic synthesised sizenanoparticles of the particles along and with not the only actual determines size of the averagemetallic sizecore but [20]. also DLS the revealssize distribution. the hydrodynamic The Gaussian size of distribution the particles data and is providednot only determines in the supplementary the average material size but (Figure also the S4). sizeThe distribution. data revealed The a bimodal Gaussian distribution distribution and data the highestis provided peak in with the thesupplementary larger fraction material was observed (Figure at S4).particle The sizedata of revealed 37.4 nm witha bimodal 83.7% intensity.distribution The and smaller the fractionhighest reveals peak with the larger the larger size of fraction nanoparticles was observedat 416.1 nm at particle with 16.3%, size whichof 37.4 proved nm with the 83.7% presence intensity. of aggregated The smaller particles fraction in the reveals sample. the Moreover, larger size the oflarge nanoparticles particle size at might 416.1 alsonm bewith because 16.3%, of which the various proved forces the of presence interaction of aggregated in the solution particles including in the van sample.der Waals Moreover, forces [20 the]. This large was particle also supported size might by also the be previous because HRTEM of the various images whereforces theof interaction variety of size in theand solution shape of including particles couldvan der be clearlyWaals forces observed. [20] Based. This onwas the also DLS supported measurements, by the the previous z-average HRTEM size of imagesthe green where synthesised the variety AuNPs of size and and citrate shape reduction of particles was could31.5 be0.5 clearly nm and observed.20.6 0.4 Based nm, onrespectively. the DLS ± ± measurements,The particles size the measuredz-average bysize DLS of the was green larger sy comparednthesised AuNPs to the HRTEM and citrat results.e reduction This is was due 31.5 to the ± 0.5circumstance nm and 20.6 that ± 0.4 the nm, measured respectively. size also The comprises particles size the bio-organicmeasured by compounds DLS was larger enveloping compared the core to theof theHRTEM AuNPs. results. However, This is thedue results to the confirmedcircumstance that that the the green measured synthesis size method also comprises using E. the elatior bio-is organiccomparable compounds to the conventionalenveloping the chemical core of the method, AuNPs. in However, terms of producingthe results confirmed small (< 50 that nm) the AuNPs. green synthesisMeanwhile, method surface using charge E. elatior is commonly is comparable quantified to the by theconventional zeta potential chemical which method, delivers in information terms of producingon the net small charge (< of50 particles nm) AuNPs. in a liquid Meanwhile, condition. surface The charge value ofis thecommonly zeta potential quantified is closely by the related zeta potentialto the suspension which delivers stability information and particle on the surface net charge coating of [particles28]. The in zeta a liquid potential condition. of the greenThe value AuNPs of thewas zeta32.0 potential0.4 mV, is closely compared related to to57 the0.2 suspension mV of the stability citrate AuNPs.and particle These surface negative coating zeta potentials[28]. The − ± − ± zetaconfirmed potential the of formation the green ofAuNPs negative was charges −32.0 ± 0.4 on themV, surface compared of the to AuNPs.−57 ± 0.2 mV of the citrate AuNPs. These negative zeta potentials confirmed the formation of negative charges on the surface of the AuNPs.2.3. Electrochemical Properties of the Modified SPCE In this study, we further applied the green synthesised AuNPs as the screen-printed carbon 2.3. Electrochemical Properties of the Modified SPCE electrode (SPCE) surface modifier by incorporating chitosan (Cs) as the polymer matrix. SPCE emerges as alternativesIn this study, to we the further conventional applied electrodes the green due synthesised to their suitabilityAuNPs as forthe the screen-printed on-site applications. carbon electrodeMeanwhile, (SPCE) chitosan surface is a non-toxicmodifier polymerby incorporat that offinger biocompatibility, chitosan (Cs) as high the mechanical polymer matrix. strength, SPCE good emergesadhesion as on alternatives electrochemical to the surfaces, conventional and subsequently electrodes enhance due to the their binding suitability of the negativelyfor the on-site charge applications.AuNPs [29]. Meanwhile, Based on Figure chitosan5, theis a peaknon-toxic current polymer of the that SPCE offer modified biocompatibility, with a mixture high mechanical of AuNPs strength,and chitosan good (SPCEadhesion/Cs/ AuNPs)on electrochemical increased bysurfac 153.95es, andµA relativesubsequently to bare enhance SPCE and the wasbinding 26% of higher the negativelycompared charge to SPCE AuNPs/Cs (213.31 [29].µ A).Based These on peakFigure increments 5, the peak could current explain of thethe enhancedSPCE modified electrocatalytic with a mixtureactivity of and AuNPs larger and surface chitosan area (SPCE/Cs/AuNPs) of AuNPs as it canincr improveeased by 153.95 the mass-transport µA relative to rate bare of SPCE electrons and 3 /4 wasbetween 26% thehigher SPCE compared and [Fe(CN)6] to SPCE/Cs− − ions, (213.31 and thereforeµA). These inducing peak increments faster electron could transfer explain kinetics. the enhancedFurthermore, electrocatalytic the larger electrochemicalactivity and larger surface-active surface area area of is AuNPs not only as beneficial it can improve for enhancing the mass- the transportsensor sensitivity, rate of electrons but also between advantageous the SPCE for and aiding [Fe(CN)6] the electron3−/4− ions, transfer and [therefore30]. Most inducing of the reported faster electronworks on transfer green synthesiskinetics. goldFurthermore, nanoparticles the larger were appliedelectrochemical as antibacterial surface-active and antifungal. area is not However, only beneficial for enhancing the sensor sensitivity, but also advantageous for aiding the electron transfer [30]. Most of the reported works on green synthesis gold nanoparticles were applied as antibacterial

Molecules 20192019, ,2424, ,x 3141 FOR PEER REVIEW 9 of 915 of 15 and antifungal. However, Bastos-Arrieta et al. [31] synthesised silver nanoparticles (AgNPs) using grapeBastos-Arrieta stalk waste et al.extract[31] synthesisedfor modification silver of nanoparticles screen-printed (AgNPs) electrodes using and grape proved stalk their waste suitability extract for formodiﬁcation sensing purposes. of screen-printed Meanwhile, electrodes Emmanuel and provedet al. [3 their2] fabricated suitability the for glassy sensing carbon purposes. electrode Meanwhile, with theEmmanuel AuNPs etsynthesised al. [32] fabricated by Acacia the nilotica glassy carbontwig bark electrode extract with and the proved AuNPs that synthesised the electrode by Acacia exhibited nilotica outstandingtwig bark extract recovery and provedresults thatand the an electrode excellent exhibited reduction outstanding ability compared recovery resultsto the andunmodified an excellent electrode.reduction ability compared to the unmodiﬁed electrode.

(a) (b) 300

250

200

150

100 Peak current (µA) current Peak

0 Bare SPCE SPCE/Cs SPCE/Cs/AuNPs

FigureFigure 5. 5. (a(a) )Cyclic Cyclic voltammograms voltammograms and and (b ()b bar) bar graph graph of bare of bare screen-print screen-printeded carbon carbon electrode electrode (SPCE), (SPCE), SPCESPCE and and chitosan chitosan (SPCE/Cs) (SPCE/Cs) and and SPCE SPCE with with a mixture a mixture of ofAuNPs AuNPs and and chitosan chitosan (SPCE/Cs/AuNPs) (SPCE/Cs/AuNPs) 3 /4 (green(green synthesised) synthesised) modified modified electrodes electrodes in in 5 5mM mM [Fe(CN)6] [Fe(CN)6]3−/4− in− phosphatein phosphate buffer bu salineffer saline (PBS). (PBS). 2.4. Detection of Copper (Cu) Ions using SPCE/Cs/AuNPs 2.4. Detection of Copper (Cu) Ions using SPCE/Cs/AuNPs The comparison of the analytical performance of the SPCE modified with citrate-capped AuNPs The comparison of the analytical performance of the SPCE modified with citrate-capped AuNPs and green AuNPs was determined based on the detection of Cu2+ ions in the solution. The detection and and green AuNPs was determined based on the detection of Cu2+ ions in the solution. The detection was carried out by the differential pulse voltammetry (DPV) analysis in six increasing concentrations and was carried out by the differential pulse voltammetry (DPV) analysis in six increasing of Cu(II) ranging from 0 to 20 ppm. In principle, the detection of Cu2+ ions using the adsorptive concentrations of Cu(II) ranging from 0 to 20 ppm. In principle, the detection of Cu2+ ions using the adsorptivestripping voltammetry stripping voltammetry (ASV) method (ASV) involves method three involves main three steps includingmain steps adsorptive including accumulation,adsorptive accumulation,electrochemical electrochemical reduction of a surface-activereduction of a complexsurface-active of the complex metal and of strippingthe metal out and [33 stripping]. The signal out of [33].the surface-confined The signal of the species surface-conf is correlatedined species to the is surface correlated concentration, to the surface through concentration, the adsorption through isotherm, the adsorptionproviding theisotherm, relationship providing between the relationship the surface andbetween bulk the concentrations surface and bulk of the concentrations adsorbate [34 of]. Hence,the adsorbateto evaluate [34]. the Hence, efficiency to evaluate of the modified the efficiency SPCE, aof control the modified experiment SPCE, was a control performed experiment using the was bare performedSPCE (without using any the modification) bare SPCE and(without the result any ismodification) provided in theand supplementary the result is materialprovided (Figure in the S5). 2+ supplementaryBased on the result, material the (Figure calibration S5). plotBased of on Cu the detectionresult, the bycalibration the bare plot SPCE of displaysCu2+ detection nonlinearity by the at barelower SPCE concentrations displays nonlinearity which support at lower that the concen electrochemicaltrations which transduction support that material the electrochemical including AuNPs transductionas an electrode material modifier including contributes AuNPs tothe as an electrochemical electrode modifier enhancement. contribute Meanwhile,s to the electrochemical Figure6 shows enhancement.the DPV responses Meanwhile, of both Figure modified 6 shows electrodes the DPV and responses the calibration of both plots modified were presentedelectrodes asand an the insert. calibrationWell-defined plots peaks were for presented Cu2+ detection as an insert. by SPCE Well-defined modified withpeaks green for Cu AuNPs2+ detection and citrate-capped by SPCE modified AuNPs withcan be green observed AuNPs at betweenand citrate-capped0.24 to 0.17AuNPs V and can be0.27 observed to 0.17 at V, between respectively. −0.24 The to − peaks0.17 V currents and −0.27 were − − − − toproportionally −0.17 V, respectively. increased The with peaks positive currents shifts were of prop peakortionally potentials increased as the concentration with positive of shifts Cu(II) of increase.peak potentialsThe DPV peakas the currents concentration were obtained of Cu(II) with increase. correction The to DPV the basepeak line currents and the were calibration obtained plots with were correctionevaluated to from the thebase peak line and currents. the calibration The results plots proved were thatevaluated the SPCE from modified the peak withcurrents. green The synthesised results provedAuNPs that performed the SPCE well modified as the electrode with green modifier synthesised and subsequently AuNPs performed enhanced well the as sensitivitythe electrode of the modifierelectrode. and Although subsequently the current enhanced produced the sensitivity by SPCE of modified the electrode. with citrate-capped Although the AuNPscurrent wereproduced slightly byhigher SPCE due modified to smaller with size citrate-capped of nanoparticles AuNPs compared were toslightly green higher synthesised due AuNPs,to smaller however, size of the nanoparticleslinearity and stabilitycompared of to green green synthesised synthesised AuNPs AuNPs, were however, observed the to linearity be greater. and stability of green synthesised AuNPs were observed to be greater.

MoleculesMoleculesMolecules 20192019, 201924, 24, x,, 3141FOR24, x PEERFOR PEER REVIEW REVIEW 10 of 101510 of 15of 15

115 (a) 115 120 (a) 120 120 0 ppm 0 (ppmb) (b) 120 0 ppm 0 ppm y = 5.3441x +y 3.1074 = 5.3441x + 3.1074 1 ppm 1 ppm 1 ppm 100 100 1 ppm 95 95 100 100 y = 4.2633xy = - 4.2633x 0.2172 - 0.2172 R² = 0.9883 5 ppm R² = 0.9883 5 ppm 5 ppm R² = 0.9978R² = 0.9978 5 ppm 80 80 80 80 10 ppm 10 ppm 10 ppm95 95 10 ppm 60 15 ppm 60 60 15 ppm 15 ppm 60 15 ppm 20 ppm 20 ppm 20 ppm

20 ppm (µA) Current 75 75 40 40 (µA) Current 40 40 Current (µA) Current Current (µA) Current 20 20 75 7520 20

0 0 0 0 55 55 0 50 10 5 15 10 20 15 20 0 50 10 5 15 10 20 15 20 ConcentrationConcentration (ppm) (ppm) 55 55 ConcentrationConcentration (ppm) (ppm) Current (µA) Current (µA) Current (µA) 35 Current (µA) 35 35 35

15 15 15 15

-5 -5 -5 -5 -0.5-0.5 -0.4 -0.4 -0.3 -0.3 -0.2 -0.2 -0.1 -0.1 -0.5 -0.4 -0.3 -0.2 -0.1 Potential (V) -0.5 -0.4Potential -0.3(V) -0.2 -0.1 Potential (V) Potential (V) Figure 6. Detection of Cu2+ ions2+ in free solution by the differential pulse voltammetry analysis in 0.1M FigureFigure 6. Detection 6. Detection of Cuof Cuions2+ ions in in free free solution solution by by the the di ffdifferentialerential pulse pulse voltammetry voltammetry analysis analysis in0.1M in 0.1M KCl/HCl buffer. (a) SPCE modified with chitosan and green synthesised AuNPs and (b) SPCE KClKCl/HCl/HCl buff er.buffer. (a) SPCE (a) SPCE modified modified with chitosan with chitosan and green and synthesised green synthesised AuNPs and AuNPs (b) SPCE and modified (b) SPCE modified with chitosan and citrate-capped AuNPs. withmodified chitosan with and chitosan citrate-capped and citrate-capped AuNPs. AuNPs. 3.3. Materials Materials and and Methods Methods 3. Materials and Methods 3.1.3.1. Materials Materials 3.1. Materials GoldGold (III) (III) chloride chloride hydrate hydrate (HAuCl (HAuCl4.xH4.xH2O)2 O)was was purchased purchased from from Sigma Sigma Aldrich Aldrich (St. (St.Louis, Louis, MO, MO, Gold (III) chloride hydrate (HAuCl4.xH2O) was purchased from Sigma Aldrich (St. Louis, MO, USA).USA). All All other other reagents reagents including including methanol methanol (MeO (MeOH),H), sodium sodium chloride chloride (NaCl), (NaCl), Folin-Ciocaltae Folin-Ciocaltae (FC) (FC) USA). All other reagents including methanol (MeOH), sodium chloride (NaCl), Folin-Ciocaltae (FC) reagent,reagent, sodium sodium carbonate carbonate (Na (Na2CO2CO3),3 sodium), sodium nitrite nitrite (NaNO (NaNO2), 2aluminium), aluminium chloride chloride (AlCl (AlCl3), sodium3), sodium hydroxidehydroxidereagent, (NaOH), (NaOH),sodium gallic carbonate gallic acid acid (QAE),(Na (QAE),2CO quercetin3), quercetin sodium (Q (QE),nitriteE), potassiumpotassium (NaNO 2chloride), aluminium chloride (KCl), (KCl), chloride hydrochloric hydrochloric (AlCl acid3), sodium acid hydroxide (NaOH), gallic acid (QAE), quercetin (QE), potassium chloride (KCl), hydrochloric acid (HCl),(HCl), nitric nitric acid acid (HNO (HNO3),3), trisodium trisodium citrate citrate (Na (Na3C36CH65HO57O), 7potassium), potassium ferrocyanide ferrocyanide (K4 (KFe(CN)4Fe(CN)6), 6), (HCl), nitric acid (HNO3), trisodium citrate (Na3C6H5O7), potassium ferrocyanide (K4Fe(CN)6), potassiumpotassium ferricyanide ferricyanide (K (K3Fe(CN)3Fe(CN)6),6 copper), copper (II) (II) chloride chloride (Cu) (Cu) and and phosphate phosphate buffer buff salineer saline (PBS) (PBS) tablet tablet werewerepotassium of analyticalanalytical ferricyanide gradegrade with with (K3 maximumFe(CN) maximum6), copper purity. purity. (II) All chlorideAll aqueous aqueous (Cu) solutions andsolutions ph andosphate suspensionsand suspensionsbuffer saline were preparedwere(PBS) tablet preparedusingwere autoclaved usingof analytical autoclaved purified grade waterpurified with (Milli-Q watermaximum RG, (Mil Millipore, li-Qpurity. RG, AllMillipore, Germany). aqueous Germany). The solutions fresh E.The elatiorand fresh suspensions(Figure E. elatior7) was were (Figurepurchasedprepared 7) was from using purchased the autoclaved wet market from purified inthe Sri wet Serdang, water market (Mil Selangor, inli-Q Sri RG,Serdang, Malaysia, Millipore, Selangor, and Germany). was Malaysia, immediately The andfresh extracted was E. elatior immediatelyupon(Figure purchase. 7)extracted was purchased upon purchase. from the wet market in Sri Serdang, Selangor, Malaysia, and was immediately extracted upon purchase. (a) (b) (c) (a) (b) (c)

Figure 7. (a) Half-bloom inflorescence of E. elatior, (b) young shoots of E. elatior and (c) cross-section ofFigure theFigure E. 7.elatior( a7.) Half-bloom(flower.a) Half-bloom inflorescence inflorescence of E. of elatior E. elatior,(b), young (b) young shoots shoots of E. of elatior E. elatiorand ( cand) cross-section (c) cross-section of theofE. the elatior E. elatiorflower. flower. 3.2. Preparation of Etlingera elatior Extract 3.2. Preparation of Etlingera elatior Extract 3.2.The Preparation E. elatior offlowers Etlingera were elatior washed Extract thoroughly and chopped. Approximately, 20 g of the choppedThe TheflowerE. elatiorE. waselatiorflowers boiled flowers were for washed1were h in washed100 thoroughly mL thoroughlyof deionised and chopped. and water chopped. Approximately,with the Approximately, addition 20 gof of 10 the mL20 chopped ofg of the methanolflowerchopped was (to initiate boiledflower forthewas 1isolation boiled h in 100 forof mLbioactive 1 h of in deionised 100 compounds). mL of water deionised The with extract thewater additionwas with then the offiltered 10addition mL using of methanolof filter 10 mL of paper (125 mm Ø, Whatman, Maidstone, United Kingdom), and the pH was adjusted to 7.5 with 1 M (tomethanol initiate the (to isolation initiate the of bioactiveisolation of compounds). bioactive compounds). The extract The was extract then filteredwas then using filtered filter using paper filter of NaOH. The extract solution was stored at 4 °C until further use. paper (125 mm Ø, Whatman, Maidstone, United Kingdom), and the pH was adjusted to 7.5 with 1 M

of NaOH. The extract solution was stored at 4 °C until further use.

Molecules 2019, 24, 3141 11 of 15

(125 mm Ø, Whatman, Maidstone, United Kingdom), and the pH was adjusted to 7.5 with 1 M of NaOH. The extract solution was stored at 4 ◦C until further use.

3.3. Determination of Polyphenolic Compounds of Etlingera elatior Extract

3.3.1. Total Phenolic Content (TPC) The concentration of total phenolic content was estimated by the Folin-Ciocalteu method with minor modiﬁcations [35]. Two hundred microlitres of the E. elatior extract was mixed with 1000 µL of the FC reagent (dilution ratio of FC reagent, 1:10 v/v) and left in the dark for 6 min. After that, 800 µL of 7.5% Na2CO3 solution were added, shaken and left in the dark for 2 h to react. The absorbance was recorded at 740 nm, and the total phenolic content of the E. elatior extract was expressed as the gallic acid equivalent (GAE/100 g) of the E. elatior ﬂower. The standard curve was constructed using a standard solution of gallic acid (Figure S1).

3.3.2. Total Flavonoid Content (TFC) The total of flavonoids content in the E. elatior extract was measured using the spectrophotometric method as described by Kamboj et al. [36]. Briefly, 1 mL of the E. elatior extract was mixed with 0.3 mL of 5% NaNO2 and left for 5 min before added with 0.3 mL of 10% AlCl3. The mixture was mixed and 6 min later 2 mL of 1M NaOH was added to neutralize the solution. The absorbance of all samples was recorded at 510 nm using the UV-Vis spectrophotometer. Total flavonoids content was expressed in a mg quercetin equivalent (QE/100 g) of the E. elatior flower. The standard curve was constructed using a standard solution of quercetin (Figure S2).

3.4. Green Synthesis of Gold Nanoparticles using the E. elatior Extract Aqua regia was prepared to clean the flask and magnetic stirring bar prior to the AuNPs synthesis. The procedure was conducted inside the fume hood with proper ventilation due to the strong acid odour. A 250-mL conical flask with a magnetic stirrer was placed in a container with ice cubes. Concentrated HCl and HNO3 were poured into the flask at a ratio of 3:1. The orange-red solution was formed, and the solution was allowed to settle for approximately 45 min. Following this, the aqua regia solution was carefully discarded, and the flask and magnetic stirrer bar were rinsed with distilled water multiple times and dried. Next, 34 mg of the HAuCl4 powder was measured in an Eppendorf tube, and 2 mL of sterile deionised water was added. The yellow solution was filtered using the dual-filter syringe nylon filter (13 mm Ø, 0.45 µm pore size, Thermo-Line, New South Wales, Australia), and 1.6 mL filtrate was added into 80 mL of sterile deionised water. The gold solution was poured into the cleaned flask and heated to 250 ◦C until bubbles formed while stirring at 70 rpm. Next, 8 mL of the E. elatior extract was added quickly to the boiled solution, and the stirring speed was increased to 200 rpm. The heating and stirring were continued for another 15 min. For the cooling step, the solution was kept stirred at 120 rpm without heating until it reached room temperature. The flask was covered with aluminium foil and kept at 4 ◦C until further use. The conventional citrate-capped AuNPs were also synthesised using the same method by replacing the green reducing agent of the E. elatior extract with 38.8 mM of Na3C6H5O7 solution.

3.5. Characterisation of Synthesised Gold Nanoparticles

3.5.1. Ultraviolet-Visible Spectroscopy UV-Vis spectroscopy was used to determine the formation and stability of AuNPs in the aqueous solution. The nanoparticle solution was diluted (1:2 dilution) with deionised water to minimise errors due to the high optical density of the solution. Next, 2 mL of the aliquot was measured in the UV-Vis spectra (Genesys 10S UV-Vis Spectrophotometer, Thermo Scientiﬁc, Waltham, MA, USA) using a Molecules 2019, 24, 3141 12 of 15

10-mm quartz cuvette. The scanning was done at a resolution of 1 nm between 200 to 900 nm with a fast mode scanning speed.

3.5.2. Fourier Transform Infrared Spectroscopy The AuNP dried samples were prepared by centrifuging the synthesised solution at 15000 g × for 15 min at 25 ◦C. The solid residual layer was dispersed in sterile deionised water thrice to remove residual biological impurities. The pure residue was then oven-dried overnight at 40 ◦C. The obtained powder was subjected to the FTIR spectrometer (Nicolet 6700, Thermo Scientiﬁc, Waltham, MA, USA) 1 measurement at a resolution of 4 cm− .

3.5.3. High-Resolution Transmission Electron Microscopy and Energy-Dispersive X-ray The surface morphology of the AuNPs was analysed using HRTEM (JEM-2100F, JEOL, Tokyo, Japan). The samples were prepared by drying a droplet of the synthesised AuNPs suspension on a copper grid (Formvar Carbon Film, FCF400-CU, 400 Mesh, EMS, Hatfield, PA, USA), overnight at room temperature. The excessed solution was removed using filter paper. Energy-Dispersive X-ray was done using the field emission scanning electron microscopy, FESEM (UHR SU8030, Hitachi, Tokyo, Japan).

3.5.4. Dynamic Light Scattering (DLS) Measurements The size and stability of the AuNPs were measured using a Zeta-sizer instrument (Malvern Nano-ZS, Malvern Panalytical, Worcestershire, United Kingdom). The measurement of the zeta potential is based on the direction and velocity of particles under the inﬂuence of the known electric ﬁeld. The material refractive index was set to n = 0.20, and material absorption, k = 3.32, using water as the dispersant.

3.6. Modiﬁcation of Screen-Printed Carbon Electrode (SPCE) using Synthesised AuNPs

3.6.1. Preparation of Modiﬁed SPCE The AuNPs solution was used as it is after the synthesis process without any puriﬁcation step or dilution. The screen-printed carbon electrode (SPCE) (Biogenes, Selangor, Malaysia) was drop-coated using the nanocomposite of AuNPs and chitosan (Cs). First, 0.5% Cs solution was prepared in 1% acetic acid and the mixture was stirred until fully dissolved. The nanocomposite was prepared by mixing 100 µL of 0.5% Cs solution with 100 µL of AuNPs solution (1:1, v/v). Then, 10 µL of the nanocomposite mixture was drop-coated onto the working electrode of SPCE. The SPCE was dried at room temperature overnight.

3.6.2. Electrochemical Characterisation of Modified SPCE The electrochemical performance of the AuNPs was tested using the cyclic voltammetry (CV) analysis. The electrochemical properties of bare SPCE were compared to the electrochemical properties 3 /4 of the AuNPs modified SPCE. The measurement was done in 5 mM of [Fe(CN)6] − − redox pair prepared in 10 mM phosphate buffer saline (PBS). The CV potential was set from 0.5 to 0.7 V with the − scan rate of 0.05 V/s. The CV measurement was done using the Potentiostat/Galvanostat µStat 400 (Metrohm DropSens, Llanera, Asturias, Spain).

3.6.3. Detection of Heavy Metal using Modified SPCE The electrochemical performance of the modified SPCE was evaluated by detection of Cu(II) in 0.1 M KCl/HCl buffer at room temperature. The measurement was done using the differential pulse voltammetry (DPV) analysis with 300 s of deposition time at 1.2 V deposition potential, 0.005 V − potential step, 0.025 V amplitude and 20 Hz frequency. The experiment was done in triplicate (n = 3). Molecules 2019, 24, 3141 13 of 15

4. Conclusions In the present work, we reported a simple and novel method to synthesise AuNPs via the reduction of aqueous HAuCl4 ions using the E. elatior extract. The developed method resulted in the formation of AuNPs with spherical, triangular and some rod shapes with an average size of 31.5 0.5 nm. ± The spectroscopic characterisation of the green synthesised AuNPs by UV-Vis, FTIR, HRTEM and EDX analyses further confirmed the formation and stability of the synthesised AuNPs. The overall results showed that this green synthesis method is comparable to the standard citrate-based method and can be considered as an alternative method. The synthesised AuNPs was further used as the electrode surface modifier which has been shown to greatly enhance the electrochemical current by enhancing the rate of electron transfer. The modified SPCE was tested for the detection of Cu(II), providing good sensitivity and linearity. The green synthesis method we proposed herein is eco-friendly, economical and can be considered for use in a large-scale production of the AuNPs by employing the abundantly available E. elatior plant.

Supplementary Materials: Figures S1–S5 and Table S1 are available in the supplementary materials. Author Contributions: Conceptualization, F.A.A. and N.N.; Methodology, N.H.A.R.; Validation, F.A.A., N.A.Y. and N.H.A.R.; Formal analysis and investigation, F.A.A.; Resources, J.S., N.A.Y., R.S. and N.N.J.; Data curation, F.A.A.; Writing—original draft preparation, F.A.A.; Writing—review and editing, J.S., R.S., N.N., N.H.A.R., N.A.Y., and N.N.J.; Visualization, F.A.A.; Supervision, J.S., R.S., N.A.Y. and N.H.A.R.; Funding acquisition, J.S. Funding: The authors would like to acknowledge the Ministry of Higher Education, Malaysia for financing the work through the Higher Institution Centre of Excellence (HICoE) grant scheme—project no.: 6369114 and Universiti Putra Malaysia for providing Graduate Research Fellowship (GRF). Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the Etlingera elatior extract and gold nanoparticles are available from the authors.

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