Introduction

The optoelectronic and physicochemical properties of nanoscale materials are size and shape dependent. Polyshaped nanoparticles, which resemble certain bacteria, can more easily infiltrate human cells, and this suggests that using the polyshape of nanoparticles can make them more effective in treating diseases [1]. The general medical applications of nanoparticles have been reviewed by Salata [2].

Gold, especially in nanosize, is widely used in emerging interdisciplinary field of nanobiotechnology [3, 4]. Their displayed novel properties have more effective and wider biomedical applications [5–11]. These properties of polyshaped gold nanoparticles (AuNP) have different behaviors and make them suitable for therapeutic utilization. Biological molecules and neoplastic cells can be readily attached to the surface of AuNP, and the plasmon resonances of such bio-adsorbed AuNP of certain shapes cause them to have greater photon capture cross-sections than those of photo thermal dyes [12]. Bio-detection sensitivity of AuNP is often associated with their physical and chemical properties depending on the shape [13, 14]. AuNP with different dimensions have been widely applied to detect biological molecules [15]. Colloidal AuNP are used to detect specific DNA sequences and single-base mutations in a homogeneous format [16].

The nanoparticle synthesis techniques fall under two categories: “bottom–up” and “top–down” approaches. With the development of top–down chemical and physical methods, the concern for environmental contaminations is heightened as the chemical procedures involved in the synthesis of nanomaterials generate a large amount of hazardous byproducts. Among the various synthesis routes, bio-based “bottom–up” protocol promises a better chance to obtain nanostructures of more homogeneous chemical composition and with fewer defects [17]. With the voracious demand for biosynthesized nanoparticles, this recently developed field offers many new routes. These include employing microorganisms, such as: Fusarium oxysporum [18], Fusarium semitactum [19, 20], Cladosporium sp. [21], and also different plants like alfalfa [22] and neem [23]. Biosynthesis of metal nanoparticles from different microorganisms is a well-known concept, but use of plants is gaining much importance because of its nonpathogenicity and offers low or nil toxic profiles. Possibility of using this process can be thought of on a large scale. Our research is focused on the microwave-exposed aqueous extracellular guava leaf extract for gold nanoparticle synthesis.

The rate of synthesis of AuNP from microwave-exposed guava leaf extract is compared to those of chemical and physical methods, and it is noted that this has an excellent time-related advantage over benign and conventional method of metal nanoparticle synthesis from microorganisms and other plants [22, 23]. Unlike microbial nanoparticle synthesis, the current method holds an advantage of being simple, carried out at ambient temperature, and synthesized with a high level of reproducibility. The stability of these gold nanoparticle suspensions stored for 30 weeks was analyzed using zeta potential instrument. The study infers that the stability of colloidal AuNP synthesized using aqueous guava leaf extract is more than that of nanoparticles synthesized from microorganisms [18–21].

Recently, Sastry et al. synthesized Au, Ag, and Au–Ag bimetallic Au core–Ag shell nanoparticles using neem (Azadicachta indica) leaf broth has consumed 2–4 h of time for the process [23]. The current method of AuNP synthesis from guava leaf extract offers a great time-related advantage as the entire process is completed within few minutes (90% of the reduction of Au3+ ions to AuNP takes place within 5 min).

Twenty-five different reported anti-malignant plants were screened including the popular anti-malignant Vinca (Vinca rosea) plant. Guava (Psidium guajava) leaf, which is also known for its anti-malignant effects, was selected after confirming its potentiality for further preparation of extract, synthesis of AuNP, and for screening. The extracellular guava leaf extract which contained a variety of flavonoids is commonly used in subtropical countries as a dietary supplement because of its various pharmacological effects like antioxidant and anti-malignant activities [24–27]. In fact, it was reported that the fermented guava leaf extract suppressed lipopolysaccharide-induced macrophage cancer cells [28]. However, only limited studies demonstrated that the plants were used to synthesize the AuNP with a preview of having synergic anti-malignant activity.

Materials and Methods

Preparation of the Reaction Solutions

Carefully weighed 10−3 M HAuCl4 aqueous solution was used for the synthesis of AuNP. For preparing the aqueous extracellular solution of guava leaves (P. guajava) for biosynthesis, 10 g of freshly collected guava leaves cut into appropriate size (∼ 5 cm × 1 cm) was taken in a 250-ml wide-neck Borosil conical flask and washed several times with deionized water. Deionized water (200 ml) was added to the flask containing washed, freshly cut guava leaves and exposed to microwave for 3 min (DAEWOO, 2.45 GHz). The resultant crude extract was filtered with Whatman filter paper no. 40. Exactly 5 ml of the filtrate was then added to 50 ml of aqueous 10−3 M HAuCl4 solution in a 250-ml Borosil flask and kept on a shaker at 27°C for the biosynthesis of AuNP.

Analysis

Periodically, aliquots of the reaction solution were removed and subjected to UV–vis spectroscopy measurements. The UV–vis spectroscopy measurements were performed on an ECIL 5704SS UV–visible spectrophotometer at a resolution of 1 nm. For the crystallinity studies, films of AuNP formed on Si (1 1 1) substrates by drop coating the colloidal nanoparticles were used for X-ray diffraction (XRD) study. The data were obtained using Siemens X-ray diffractometer (Japan), operated at 30 kV and 20 mA current with Cu Kα (l = 1.54 Å) radiation.

The morphology of the AuNP was examined using field emission scanning electron microscopy (FESEM, FEI Nova nano 600, Netherlands), and for this the images were operated at 15 kV on a 0° tilt position. The transmission electron microscopy (TEM) image of the sample was obtained using Technai-20 Philips transmission electron microscope operated at 190 keV. Atomic force microscopy (AFM) images were collected under ambient conditions on a Veeco Innova scanning probe microscope. Etched Si nanoprobe tips (RTESPA-M) were used for the same. AuNP powder sample was prepared by centrifuging the synthesized AuNP solution at 10,000 rpm for 15 min for Fourier transformed infrared radiation (FTIR) Spectroscopy measurements. The solid residue layer which contains AuNP was redispersed in sterile deionized water three times to remove the unattached biological impurities. The pure residue was then dried perfectly in an oven overnight at 65°C. The thus obtained powder was subjected to FTIR measurements carried out on a Perkin-Elmer Spectrum-One instrument at a resolution of 4 cm−1 in KBr pellets.

Zeta potential of the AuNP colloidal solution stored up to 30 weeks in ambient conditions was sonicated and measured using a PALS Zeta Potential Analyzer ver. 3.54 (NCL, Pune).

Results and Discussion

In this paper, we stress on the use of microwave-exposed aqueous extracellular guava leaf extract for the synthesis of gold nanoparticles (AuNP) from aqueous HAuCl4 solution. (i.e., through the reduction of Au3+ to Au0). The initial color of the solution after the addition of microwave-exposed aqueous guava leaf solution to aqueous HAuCl4 solution was colorless. The intensity of the color of reaction mixture changes to dark purple with in 5 min. This shows that the reaction medium behaves with time kinetics; the same is shown in Fig. 1. The color developed in the solution as a result of the formation of AuNP. The same is observed with a surface plasmon resonance (SPR) peak in the UV–visible spectroscopy. This important observation indicates that the reduction of the Au3+ ions takes place extracellularly. Color of the AuNP found to be dark purple in water is due to excitation of the SPR in metal nanoparticles [29]. The biosynthesis reaction accelerates with the time, and the intensity of color of the reaction mixture increases rapidly. The process of biosynthesis is carried out at ambient environmental conditions, and the total reaction is completed within few minutes. It is observed that the surface plasmon peak occurs at 505 nm; the wavelength peak is shown in Fig. 2. The broadening and slight shifting of the SPR is probably due to the dampening of the surface plasmon resonance caused by the change in the refractive index of the surrounding medium and also due to the modification of the size and shape of the AuNP in colloidal solution. The reduction of the metal ions occurs very rapidly, and more than 90% of the reduction of Au3+ ions will be completed within 5 min. This is the fastest method of biosynthesis in comparison to the earlier conventional studies with other microorganism- [18–21] and plant-mediated [22, 23] synthesis. The time required for the conventional synthesis of AuNP from other plants was 2 to 4 h and from different microorganisms was 24 to 120 h and thus are rather slow.

Fig. 1
figure 1

Color intensity of AuNP. The intensity of color of the solution containing aqueous 10−3 M HAuCl4 treated with microwave-exposed guava (P. guajava) leaf extract increases steadily as a function of reaction time.

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Fig. 2
figure 2

UV–Visible spectra recorded as a function of time (λ max = 505 for AuNP). For brevity, data corresponding to all spectra are not plotted.

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Figure 3 shows the XRD pattern suggesting the crystalline nature of the AuNP synthesized from microwave-exposed aqueous extracellular guava leaf extract. The intense diffraction peaks due to AuNP are clearly observed at (111), (200) (220), and (311). These agree well with the reported standards JCPDS file no. 04-0784. This study infers the crystallinity of the AuNP obtained during biosynthesis. The mean particle diameter of AuNP was calculated from the XRD pattern according to the line width of the (1 1 1) refraction peak using the following Scherrer's equation:

Fig. 3
figure 3

XRD pattern of AuNP.

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$$ D = \frac{{K\lambda }}{{{\beta_{{\raise0.5ex\hbox{$\scriptstyle 1$}\kern-0.1em/\kern-0.15em\lower0.25ex\hbox{$\scriptstyle 2$}}}}\cos \theta }} $$

The equation uses the reference peak width at angle θ, where α is the X-ray wavelength (1.5418 Å), β 1/2 is the width of the XRD peak at half height, and K is a shape factor which is 0.9 for spherical particles. The calculated average particle size of the Au was found to be 27 nm, which was also in line with the observation of the TEM results discussed later. The slight deviation of the band from 150 to 250 seen was due to organic moiety [19]. The energy-dispersive X-ray analysis study (EDAX) verifies the crystallinity of the particles AuNP, shown in Fig. 4.

Fig. 4
figure 4

EDAX graph of metallic AuNP.

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Figure 5 shows the FESEM of synthesized AuNP on nanometric scale. The nanoparticles were uniformly distributed and are found to have formed in an aggregated state. On careful observation, AuNP are found to be surrounded by a thin layer of bio-moieties (mostly flavonoids) which seem to be responsible for reducing Au3+ ions to AuNP. Most of the particles are spherical in shape and monodispersed in nature with diameters in the range of 27 ± 3 nm. The triangular and hexagonal shaped nanoparticles comparatively are in bigger size and probably have contributed for size variation. The same is reconfirmed with drop-coated TEM grids. It can be understood that the absolute monodispersity can be made possible by varying the other environments like pH of the extract, concentration of the aqueous salt solution, temperature, etc. of the reaction. The studies are under way.

Fig. 5
figure 5

FESEM images of AuNP. The inset shows the homogeneity of AuNP showed at 1 μm scale.

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The size and shape distribution histogram obtained for 1,107 nanoparticles is shown in Fig. 6. This histogram is prepared from the statistical data provided along with FESEM images. From this figure, it is observed that above 75% of the particles are in 24–29 nm range, and there is a marginal variation in the particle size. In this range, 95% of the particles are in spherical shape, and the remaining are of triangular shape. In this range, very rare pentagonal structures are also observed. In the 30–35 nm range, hexagonal nanoparticles are in high concentration, and the remaining are in triangular shape. Surprisingly, in this range, no spherical shaped nanoparticles are observed. At the lower range from 18 to 23 nm, triangular nanoparticles are also present in moderate concentration (∼10%), and the remaining portion is of spherical nanoparticles only. Rod-shaped particles are seen in the lower range of 7–12 nm, but overall contribution of these shapes to the total number of nanoparticles taken for analysis is negligible (0.2%).

Fig. 6
figure 6

Particle size distribution histogram of AuNP using FESEM measurements.

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Figure 7 shows the typical bright-field TEM image of the biosynthesized AuNP. TEM technique was employed to visualize the size and shape of the AuNP formed. The TEM and FESEM images suggest that the particles are almost monodispersed and polyshaped. The TEM image infers that there are few triangular and hexagonal shaped particles, and the remaining are spherical shaped AuNP.

Fig. 7
figure 7

TEM images of AuNP.

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AFM is becoming an important biophysical technique for studying the morphology of nanoparticles and biomolecules [30]. The understanding of the functionalization of the AuNP synthesized from microwave-exposed aqueous extracellular extract of guava leaves is further confirmed by AFM measurements. The three-dimensional study of bio-functionalized AuNP is done by AFM and further compared with FESEM and TEM results. The AFM measurements were made using the tapping mode developed especially for studying bio-functionalized samples. Figure 8 shows typical, medium-scale AFM image (5 × 5 µm) of the bio-functionalized organic layer which consists of rich concentration organic moieties at the surface. From the topographical view, it is evident that most of the nanoparticles are in spherical and regular shapes. The root mean square value for roughness of the sample is only about 27 nm. The results as noted above in FESEM images of Fig. 4 and the TEM images of Fig. 5 are quite agreeable to the observations made under AFM results, and further studies are required to know the clear dimension of the thickness of the moieties covered on the nanoparticles. The particle size in the AFM images, ∼40–60 nm, is somewhat larger than the value determined from TEM measurements (∼25–30 nm). This magnification is attributed to the convolution of true particle size with that of the AFM tip (increase in size and shape of AuNP observed in AFM may be influenced by the effect of tip-sample convolution). The individual polyshaped AuNP are clearly illustrated using different microscopic techniques in Fig. 9

Fig. 8
figure 8

AFM images of AuNP.

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Fig. 9
figure 9

Shapes of AuNP observed under different microscopic techniques.

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The probable pathway of biosynthesis was studied using Fourier transformed infrared spectroscopy. Figure 10 shows that FTIR peaks at 1,704, 1,607, 1,385, 1,197, and 1,082 cm−1 represent the different functional groups of the adsorbed biomolecules on the surface of the AuNP. Absence of amide I and amide II peaks confirms the denaturing of proteins/enzymes in the guava extract after the microwave exposure. This also suggests the influence of other organic moieties in the synthesis and stabilization of AuNP. The probable pathway of bioreduction is shown as schematic diagram in inset of Fig. 10. We can presume that the flavonoids [31] and terpenoids [32], which are abundant in guava leaves, show characteristic absorption peaks that appear to be responsible for accelerated reduction and capping process. Terpenoids are poorly water soluble and hence may not be among prime moieties involved in the reduction process. The IR absorptions peaks (cm−1) are noted as follows: 1,197 and 1,082 (–O–), 1,385 cm−1 (geminal methyl group, 1,607 (C=C, aromatic), 1,704 (α,β unsaturated ketone and esters). The peaks observed suggest the presence of flavonones adsorbed on the surface of the AuNP. It can be understood that the flavonoids could be adsorbed on the metal surface by interaction with carbonyl groups or π-electrons. The internal conversion mechanism of ketone group to carboxylic acid in flavonoids may influence the Au3+ ion reduction. The study is under way. The nanoparticles are almost of spherical shape and dispersed in nature with diameters in the range of 27 ± 3 nm. The same is reconfirmed with drop-coated TEM grids.

Fig. 10
figure 10

FTIR spectra of dried, ultracentrifuged, and separated AuNP powder in KBR pellets. Inset shows the probable pathway of extracellular biosynthesis of AuNP from bioreducing agents of guava (P. guajava) leaf extract.

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With the advent of nanobiotechnology, the study of colloids has become very important, especially in usage of these colloidal solutions in drug delivery wherein stable dispersions are required. The zeta potential of 30 weeks stored colloidal solution of bio-stabilized AuNP was −30 mV, which suggests the fair redespersibility of the particles which is shown in Fig. 11 [33]. The increase in the particle size of the AuNP as mentioned in the figure may be because of the agglomeration of the colloids or the increased organic capping on the surface of the AuNP. The entire exercise infers that AuNP synthesized and stored for 30 weeks at room temperature were much more stable than the nanoparticles synthesized from the other biological routes. Bio-functionalization, i.e., the nature of capping conferring the stability of the AuNP obtained by this method, is quite important, as this method produces extremely stable metallic nanoparticles.

Fig. 11
figure 11

Zeta potential studies of functionalized AuNP synthesized using aqueous guava extract with aqueous AgNO3 solution after 30 weeks.

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Conclusion

Rapid synthesis and excellent reproducibility of gold nanoparticles through this method has the time-related advantage in comparison with microbial synthesis and avoids all lengthy, laborious processes and also hygienic complications associated with them. Using the clean-green chemistry involved in this way, we can think of the industrial scaling of nanoparticles. This field of gold nanoparticle biosynthesis is leading to new avenues in material science, chemistry, nanobiotechnology and biotechnology. In future, it would be significant to understand the clear mechanism of biosynthesis and to technologically engineer the nanoparticles in order to achieve better control over size, shape, and absolute monodispersivity.