1 Introduction

Zinc oxide is an important inorganic material due to its low cost, large band gap (3.37 eV), large excitation binding energy (60 MeV), and luminescent properties [1]. It is widely used in many applications, such as catalyst, transparent conducting electrodes of solar cells, gas sensor, surface acoustic devices, flat panel displays, chemical/ biological sensors, UV lasers, and antimicrobial materials [2–7]. Nowadays, various inorganic metal oxides such as TiO2, CuO, CaO, AgO, SiO2, Fe2O3, CeO2, MgO, PbS, and ZnO are used as considerable antimicrobial agents in biological studies [8–11]. However, the use of organic material as capping agents in metal oxides will prove more powerful technique to improve the antimicrobial activities. Also, the use of organic moieties as capping agents is more stable than ordinary metal oxide nanomaterials. The ZnO nanoparticles (ZnO nps) are benign to human being as well as to environment; therefore, these materials can be called as eco-friendly materials. Indeed, the zinc element is an essential constituent for cell growth as it inhibits bacterial enzymes, thiol peroxidase, and glutathione reductase as protective enzymes [12]. The size as well as the colloidal stability of the nanoparticles can be controlled by using suitable capping agents. Particularly, the amino acids, such as histidine and cysteine, are favorable chelators with zinc and cadmium elements which act as promising capping agents for ZnS, CdS, etc. The synthesis of ZnO nps with amino acid capping agents which gives the perspective of structure, thermal, optical, and antimicrobial properties has still not been studied. It is found that among various amino acids which are used for capping, the l-histidine is the most effective in reducing the size of nanoparticles as evidences from SEM, XRD, and absorption patterns. In that respect, in the present work, we have carried out antibacterial activities of histidine-capped zinc oxide nanoparticles in different concentrations by using agar well diffusion method.

Nonetheless, tremendous interesting structures of zinc oxide nanomaterials are reported like ZnO nanostructures including nanotetrapods, nanomultipods, shuttle-like, combs-like nanorods, nanobridges nanosheets, nanoflowers, nanowires, nanoneedles, nanoribbons, nanochains, nanowalls, nanorods, and nanonails [13–16]. These structures are expected to have potential applications in building functional nanoelectronic devices. Control of the particle shape is of considerable interest for nanostructures material synthesis because of its electrical and optical properties of nonmaterial which depends on both size and shape of the particles [17].

Recently, many methods have been developed to synthesize zinc oxide nanocrystals including vapor phase growth, vapor-liquid-solid process, electrophoretic deposition, sol-gel process, homogeneous precipitation [18–23]. The methods can be used to prepare thermodynamically stable and metastable states including novel materials that cannot be easily formed from other synthetic routes [24]. However, expensive raw materials, complex process control, and sophisticated equipment are often needed, and this is unfavorable for large-scale synthesis. As a better alternative route, solvothermal methods have proven to be appealing because of their low growth temperature and good potential for scale up. The solvothermal process can be used to prepare many geometries including thin films, bulk powders, single crystals, and nanocrystals. Besides, the morphologically (sphere (3D), rod (2D), or wire (1D)) formed crystals are controlled by manipulating the solvent supersaturating, chemical of interest concentration, and kinetic control.

In this paper, we have presented simple method of regulating the shape and average size of histidine-capped ZnO nps by using solvothermal method. The synthesized ZnO nps were characterized by various spectroscopic techniques in order to estimate the structures. Further, the main aim of the present work is to describe antibacterial activities at different concentrations by agar well diffusion method against human bacterial pathogen such as Staphylococcus aureus, Escherichia coli, Klebsiella sp., Enterococcus faecalis, and Pseudomonas aeruginosa. The obtained antibacterial results clearly suggest that due to the presence of metal oxide nanoparticles which causes to damage the cell wall membrane of Gram-positive and Gram-negative bacteria. Also, it is well-known that nanoparticles have a distinct advantage over conventional chemical antimicrobial agents. In general, the chemical agents responsible for antimicrobial mechanism depend on specific binding of the surface of the agents inside the microorganisms. The antibacterial activity of histidine-capped ZnO nps can be considered to be the best effective as the zinc oxide nanomaterials generates hydrogen peroxide from its surface which inhibits the growth of the cells due to its relative permeability toward bacterial cell membrane. To the best of our knowledge, this is the first laboratory study on zinc oxide with histidine-capped nanoparticles prepared by solvothermal method and their bactericidal activity against the pathogenic bacteria using standard antimicrobial method.

2 Materials and Methods

2.1 Chemicals

All the chemicals were used of analytical grade and without purification. These were purchased from Merk Germany. The pathogens Staphylococcus aureus, Escherichia coli, Klebsiella sp., Enterococcus faecalis, and Pseudomonas aeruginosa were obtained from the Pathology and Diagnosis laboratory Department of Microbiology, Seth Kesarimal Porwal College, Kamptee, India. The strains were maintained on nutrient agar slants at 4 °C. The antibacterial activity of the histidine-capped ZnO nps was performed by using well diffusion method. About 20 ml of sterile molten Muller Hinton agar (HiMedia Laboratories Pvt. Limited, Mumbai, India) was poured into sterile petriplates. And, four well-known antibiotics viz. Amikacin (30 mcg), Ciprofloxacin (5 mcg), Gentamicin (30 mcg), and Norfloxacin (10 mcg) were obtained from Hi-Media Pvt. Ltd. Bombay.

2.2 Synthesis of Histidine-Capped ZnO nps

In this article, the synthesis of nanosized ZnO nps was carried out by dissolving 0.2 M histidine in absolute ethanol. To this solution, 0.1 M zinc acetate dihydrate and 0.1 M NaOH were added. After mixing all the three constituents in the beaker, it was transferred to polypropylene vessel, sealed and heated in temperature-controlled autoclave at 90 °C. After 12 h, a white precipitate was centrifuged and washed several times to remove undesirable impurities with double distilled water, absolute ethanol, and acetone. The precipitate was then dried at 60 °C and stored in vacuum desiccators for 24 h. In order to determine the structure and size, the various characterizations based on spectral techniques such as XRD, EDS, SEM, FT-IR, UV-visible, photoluminescence, and TG/DTG/DTA were analyzed; further, their antibacterial activities were evaluated using different human pathogens by agar well diffusion method.

2.3 Characterization of Histidine-Capped ZnO nps

2.3.1 X-ray Diffraction Measurements

The crystal structure of the sample was characterized by Bruker AXS D8 Advance X-ray diffractometer using CuKα radiation. The crystallite domain size of X-ray diffraction peaks was obtained from the width of resulted peaks. All the diffraction peaks agreed with JCPDS 36-1457. The crystalline size of the particles has been estimated by Debye-Scherrer formula.

2.3.2 Energy Dispersive X-ray Analysis

The qualitative elemental analysis of the sample was studied with the help of JEOL Model JED-2300 Energy dispersive X-ray analysis (EDAX). This technique is also useful to find out the percentage of element in a synthesized sample. This is the powerful technique to identify an element contained in the samples.

2.3.3 Scanning Electron Microscopy

The morphology, size, and shape of ZnO nps sample were examined by JEOL model JSM-6390LV scanning electron microscopy whose maximum magnification is ×3,000,000. The SEM technique is vitally important to identify the nanosize of synthesized samples.

2.3.4 UV-vis and Photoluminescence Spectroscopy

UV-vis measurements were made by a Varian, Cary 5000 UV-vis-NIR Spectrophotometer. The PL measurement of excitation and emission was recorded on the Shimadzu RF5301PC spectrofluorophotometer fitted with a sensitive photomultiplier tube. This spectrofluorophotometer at the room temperature provides corrected emission and excitation spectra in the 220–400 and 300–700 nm ranges, respectively.

2.3.5 Thermal Properties

The thermal analysis of the ZnO nps sample was recorded on PerkinElmer STA 6000 at the heating rate 4 °C/min under nitrogen atmosphere at temperature range 35–210 °C. Presence of lattice water in ZnO nps sample was confirmed by its thermograph. Further, the thermal degradation behavior of organic capping agent (histidine) at each step was determined by thermogravimetric (TG) and differential thermal analysis (DTA).

2.4 Bacterial Pathogens and Growth Conditions

The antimicrobial analysis was carried out at the Pathology and Diagnosis laboratory Department of Microbiology, Seth Kesarimal Porwal College Kamptee, Rashtrasant Tukadoji Maharaj Nagpur University Nagpur (India). The strains were maintained on nutrient agar slants at 4 °C.

2.4.1 Preparation of Inoculum

Stock cultures were maintained at 4 °C on nutrient agar. Active cultures were prepared by transferring a loopful of culture from the stock to test tubes of Mueller-Hinton broth (MHB) that were incubated for 24 h at 37 °C. The cultures were diluted with fresh MHB to achieve optical densities corresponding to 0.5, i.e., 105 to 106 CFU/ml using MacFarland’s standard.

2.5 Antibacterial Assay

2.5.1 Agar Well Diffusion Method

The antibacterial activity of the histidine-capped ZnO nps was performed by using well diffusion method. About 20 ml of sterile molten Muller Hinton agar (HiMedia Laboratories Pvt. Limited, Mumbai, India) was poured into sterile petriplates. Triplicate plates were swabbed with the overnight culture (108 cells/ml) of pathogenic bacteria viz., Pseudomonas sp., Enterobacter sp., Klebsiella sp., E. coli, and S. aureus. The solid medium was gently punctured with the help of cork borer to make a well. Finally, the nanoparticle samples (20, 40, 60, 80, 100 μg/ml) were added from the stock into each well and incubated for 24 h at 37 ± 2 °C. After 24 h, the zone of inhibition was measured and expressed as millimeter in diameter.

2.5.2 MIC

About 500 μl of different concentrations (2.5, 5, 10, 15, and 20 μg) of chosen nanoparticles were prepared with DMSO and mixed with 450 μl of nutrient broth to grow with 50 μl of 24 h old bacterial inoculums and allowed to grow overnight at 37 °C for 48 h. Nutrient broth alone served as negative control. The minimum inhibitory concentration (MIC) was the lowest concentration of the nanoparticles that did not permit any visible growth of bacteria during 24 h of incubation on the basis of turbidity [25].

3 Results and Discussion

3.1 Preparation of ZnO nps

The solvothermal method is more suitable for the production of ZnO nps. This method has been widely accepted by various researchers. The synthesis of ZnO nps by using capping agents or protective agents is an interesting work, as it enhances the novel properties of metal oxide nanoparticles. Up to this period, very less work has been done by using capping agents. Hence, we have chosen this technique for the production of ZnO nps by taking histidine as protective agents with excellent stability. In the present article, we have prepared 0.2 M histidine solution in absolute ethanol in 100-ml beaker. Thereafter, 0.1-M solution of zinc acetate dihydrate and 0.1 M sodium hydroxide in different two beakers were prepared. Then, these two solutions of zinc acetate dihydrate and sodium hydroxide were added to the previous beaker containing histidine solution. After mixing solutions, it was transferred to polypropylene vessel where it was sealed and heated in temperature-controlled autoclave at 90 °C. After 12 h, a white precipitate was centrifuged and washed several times to remove undesirable impurities with double distilled water, absolute ethanol, and acetone. The precipitate was then dried at 60 °C and stored at vacuum desiccators.

Furthermore, more attention has been paid to evaluate the antimicrobial investigation of nanoparticles. The antibacterial activities of histidine-capped ZnO nps were tested against human bacterial pathogen such as Staphylococcus aureus, Escherichia coli, Klebsiella sp., Enterococcus faecalis, and Pseudomonas aeruginosa by agar well diffusion method. Also, the antibacterial activity of ZnO nanoparticles (20 to 60 μg) was compared with four well-known antibiotics viz. Amikacin (30 mcg), Ciprofloxacin (5 mcg), Gentamicin (5 mcg), and Norfloxacin (10 mcg). In order to determine the structure and size of the prepared sample, the investigation of antibacterial activities of sample was characterized by various spectroscopic techniques such as XRD, SEM, EDS, FT-IR, UV-visible, photoluminescence, and TG/DTG/DTA studies. These various techniques have been discussed systematically in the article in order to know the structure and sizes.

3.2 Structural Analysis of ZnO nps by PXRD

From systematic investigation, PXRD curve of histidine-capped ZnO structures was performed by using several techniques. The structural compositions of the prepared samples were confirmed by PXRD studies. The PXRD spectra were recorded by using CuKα and PXRD pattern of histidine-capped ZnO (Fig. 1). The calculated lattice parameters were also in agreement with the reported values. The intense diffraction peaks were observed at 31.67°, 34.41°, 36.19°, 47.48°, 56.49°, 62.80°, 67.85°, and 69.01°, which correspond to the (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 0 3), (1 1 2), and (2 0 1) planes, respectively. The intense peaks in the PXRD pattern of these samples clearly show that the synthesized materials were ZnO with hexagonal wurtzite structure, and all diffraction peaks agreed with JCPDS 36–1457. The crystalline size of the particles has been estimated by Debye-Scherrer formula.

Fig. 1
figure 1

PXRD pattern of histidine-capped ZnO nps

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$$ D = \frac{K\lambda }{\beta cos\theta} $$

where

K :

is a constant equal to 0.89

λ :

is the wavelength of XRD radiation (λ = 0.154095 nm)

β :

is the full width at half maximum

θ :

is the diffraction angle.

A defined line broadening of the diffraction peaks is an indication that the synthesized materials are in nanometer range. The average crystalline size was calculated to be 22 nm using the diffraction intensity of (101) peak using Debye-Scherrer equation.

3.3 SEM and EDAX Analysis

The SEM image of histidine-capped ZnO nps has been displayed in the present article (Fig. 2). From Fig. 2, it is clear that the morphologies of histidine-capped ZnO nps showed as a beads shape as well as it appeared like stack of globule droplet. The bigger size of ZnO nps which appears like a droplet is due to the mishmash of various single beads. Due to high level of network formation, a big aggregation of the particles has taken place. The presence of l-histidine has brought significant surface modification of ZnO, resulting in the formation of nanocrystals of smaller size. From Fig. 2, one can see that the size of nanoparticles increases as the coagulate to forms bigger particle; therefore, ZnO nps selected for bacterial activity as they showed the smallest sized range and agglomeration. The smaller the size of nanoparticles, more will be the antibacterial activity [26]. Moreover, the activity of zinc oxide nps will be enhanced after capping the organic moiety. Also, it is known that, the activity depends on the larger surface area as well as concentration of the histidine-capped zinc oxide nps.

Fig. 2
figure 2

SEM image of histidine-capped ZnO nps

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Furthermore, the composition of synthesized nanoparticles was characterized by energy dispersive X-ray analysis. EDAX of sample (Fig. 3) has confirmed the existence of carbon, oxygen, hydrogen, and nitrogen of histidine along with zinc and oxygen of ZnO nps. The EDAX spectra are confirming that capping has been effective, and besides, zinc and oxygen are also indicating the formation of l-histidine-capped ZnO nps.

Fig. 3
figure 3

Energy-dispersive X-ray analysis of histidine-capped ZnO nps

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3.4 Infrared Spectroscopy

In addition, histidine-capped ZnO nps was characterized by the FTIR spectroscopy (Fig. 4). The FTIR spectroscopy was carried out in the range of 400–4000 cm−1. Zinc oxide generally gives absorption bands in fingerprint region, i.e., below 1000 cm−1 arising from interatomic vibrations. The broad absorption peak at 3457 cm−1 corresponds to the –OH of water molecules adsorbed on the surface of nanoparticles. The peak at 3457 cm−1 is due to NH2 stretching, peak at 1178 cm−1 corresponds to wagging of NH2, peak at 1059 cm−1 corresponds to rocking of CH2 group, and peak 1692 cm−1 corresponds –COOH group of histidine. The strong peaks that appeared near 2918 and 1580 cm−1 were assigned as zinc-coordinated –NH3 + and COO− group of the histidine amino acid. However, peaks at 2918 cm−1 also overlap with that for the C-H stretching bands in histidine molecule. To remove uncoordinated or unreacted histidine molecule, the centrifuged white solid was quickly washed several times with cold water and alcohol solution. These observations confirm the presence of capping agent in capped ZnO nps.

Fig. 4
figure 4

FTIR spectra of histidine-capped ZnO nps

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3.5 Optical and Photoluminescence Properties

The UV-vis reflectance study of histidine-capped ZnO nps is briefly described in the present article (Fig. 5). The sudden decrease of reflectance at a particular wavelength indicates that all the particles are uniformly distributed in the sample [27]. The direct band gap energy (Eg) for the histidine-capped ZnO nps (Fig. 6) was determined by extrapolating the linear of the (αhυ)2 curves vs. photo energy (hυ) and using Tauc equation.

Fig. 5
figure 5

UV-vis reflectance spectra of histidine-capped ZnO nps

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Fig. 6
figure 6

Band gap energy of histidine-capped ZnO nps

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$$ {\left(\alpha h\upupsilon \right)}^2=A\left(h\upupsilon - Eg\right) $$

where

Α :

Optical absorption coefficient

hυ:

The photon energy

Eg:

The direct band gap

A :

Constant

The direct band gap was found to be 3.4 eV which is higher than that of 3.37 eV reported in the literature. The direct band gap energy increases with decreasing particle size due to quantum size effects [27]. The room temperature photoluminescent excitation and emission spectra of histidine doped ZnO nps are shown in Fig. 7. From Fig. 7, the excitation peak was found to be at λ exc = (362 + −) nm. The inset in Fig. 7 shows corresponding photoluminescent excitation spectra (λ em = 540 nm) of histidine-doped ZnO nps. These excitation peaks correspond to band to band transition which confirms the blue shift in the band gap of ZnO nps [28].

Fig. 7
figure 7

Room temperature photoluminescence spectra of histidine-capped ZnO nps

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3.6 Thermal Analysis

The thermal degradation curve (TG/DTG) of histidine-capped ZnO nps is reported in Fig. 8 and interpreted concisely. The initial and final decomposition temperature and total mass losses for each step in the thermal decomposition of nps were investigated by using thermal analysis at a heating rate of 4 °C/min under nitrogen atmosphere over temperature in the range of 35–210 °C. The peak temperatures, and initial and final decomposition temperatures were identified at a distinct step by DTG analysis, whereas the DTA method was used to evaluate the endothermic or exothermic weight loss.

Fig. 8
figure 8

a TG-DTG and b TG-DTA curve of histidine-capped ZnO nps

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Figure 8 displays the thermal decomposition behavior of histidine-capped ZnO nps via two-step decomposition process. The structural transformation was observed by TG curves that were supported by DTG and DTA studies. The first step of decomposition in the range of 35–100 °C associated with T DTG peak at 78 °C corresponding to the mass loss of lattice water, and significantly, these values were further supported by elemental analysis and IR frequencies. In general, water of hydration may be considered either as coordinated water or lattice water, and the release of lattice water was reported with good agreement [29–32]. The low temperature range corresponding to this transformation indicates the presence of crystallization of water. The second step decomposition of ZnO nps showed the mass loss at range of 100–210 °C associated with T DTG peaks at 120 °C and 155 °C and weak endothermic T DTA peak at 122 °C due to the mass loss of histidine molecule, and this peak point indicates the initial and half decomposing temperature. This temperature range was in good agreement for the release of organic moieties. After thermal degradation of histidine molecules, a straight graph was found due to the formation of zinc oxide which demonstrated the a small thermal stability of nps. Hence, thermal analysis technique suggested the presence of histidine-capped, water crystallization and thermal stability of ZnO nps.

3.7 Antibacterial Activity

The antibacterial activity of histidine-capped ZnO nps in different concentrations (20–100 μg) was quantitatively assessed on the basis of zone of inhibition (Fig. 9 and Table 1). Among the Gram-negative bacteria tested, P. aeruginosa shows strong inhibition by histidine-capped ZnO nanoparticles not only at higher concentration of 28 mm but also at low concentration of 20 mm. Similarly, Klebsiella pneumoniae at 14 mm and E. coli at 15 mm were moderately inhibited by ZnO nps. Besides, Gram-positive bacteria was tested; S. aureus (21 mm) exhibits a maximum zone of inhibition at 100 μg of histidine-capped ZnO nanoparticles, whereas Enterobacter sp. shows intermediate zone of inhibition at lower concentration (20 μg). The present study indicates that histidine-capped ZnO nps exhibits a strong antibacterial activity against the entire pathogens even at lower concentration.

Fig. 9
figure 9

The antibacterial activity of histidine-capped ZnO nps in different concentrations (20–100 μg)

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Table 1 The antibacterial activity of histidine-capped ZnO nps in different concentrations (20–100 μg)
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After the comparison of the values of zone of inhibition with four different well-known antibiotics, viz. Amikacin (30 mcg), Ciprofloxacin (5 mcg), Gentamicin (30 mcg), and Norfloxacin (10 mcg), it was found that ZnO nps are good inhibitor of bacterial growth and show almost similar antibacterial activity as that of four investigated antibiotics toward all the tested human pathogens. P. aeruginosa exhibits resistance to Amikacin and Gentamicin but inhibited by Norfloxacin (24 mm) (Table 2). Similarly, S. aureus exhibits high sensitivity against Norfloxacin (30 mm), but minimum zone of inhibition obtained against Amikacin and Gentamicin. This shows that even at the presence of Amikacin and Gentamicin resistant, P. aeruginosa was strongly inhibited by ZnO nps at concentration (40 μg). Similarly, Amikacin-resistant S. aureus and Enterobacter sp. were inhibited by ZnO nps at low concentration (20 μg).

Table 2 Antimicrobial activity of standard antibiotics against various human pathogenic bacterial strains
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Furthermore, the MIC revealed that the histidine-capped ZnO nps shows sensitivity at the concentration of 10 μg/ml against E. coli, and Klebsiella sp. and P. aruginosa at 15 and 15 μg/ml against S. aureus (Table 3). This activity might be due to the size, surface morphology, particle morphology, and structure of the nanoparticles [33].

Table 3 MIC (μg/ml) of ZnO nps against human pathogens
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The smaller size of nanoparticles showed higher antimicrobial activity [26]. However, the activity depended on the larger surface area and concentration of histidine-capped ZnO nps, while the particle shape and crystalline structure would bring a little effect on its antimicrobial activity [33]. However, the antimicrobial mechanism of histidine-capped ZnO nps would be mainly due to physical blockage by transport channels and damage the cell membrane of bacteria due to abrasion [34].

The present study also demonstrated that S. aureus is highly sensitive to histidine-capped ZnO nps than E. coli, which is in accordance with Reddy et al. [35]. In the case of Gram-negative organisms, histidine-capped ZnO nps have to cross the outer membrane along with the thin peptidoglycan, whereas Gram-positive bacteria have thick peptidoglycan layer (30 mm) and the outer membrane [36]. The results obtained in this work clearly indicated that the presence of nanoparticles leads to the damages of the cell wall membrane of Gram-positive and Gram-negative bacteria. It is well-known that nanoparticles have a distinct advantage over conventional chemical antimicrobial agents. In general, the chemical agents responsible for antimicrobial mechanism depend on specific binding of the surface of the agents inside the microorganisms [37].

4 Conclusions

Histidine-capped ZnO nps were efficiently synthesized by solvothermal method and characterized by PXRD, SEM, EDAX, FT-IR, TG/DTG/DTA, and UV-visible spectroscopy. The average crystalline size was calculated to be 22 nm according to half width of (101) diffraction peak using Debye-Scherrer formula. The UV-visible reflectance spectroscopy, the sudden decrease of reflectance at a particular wavelength, indicates that the all particles are uniformly distributed in the sample. It was found that capping with l-histidine is effective in modifying the size and size distribution and also the PL emission characteristics of histidine-capped zinc oxide nps as compared to the pure ZnO. The effective capping with the l-histidine has been confirmed from the FTIR, thermal, and EDAX studies.

The antimicrobial activities showed that histidine-capped ZnO nanoparticles have better bacteriostatic activity against all the tested pathogens. The zone of inhibition observed against Gram-positive and Gram-negative bacteria suggests that histidine-capped ZnO nanoparticles may be the promising antibacterial agents. The antibacterial activity increases with the decreasing particle size and increasing concentration of ZnO nps. l-Histidine-capped ZnO nanoparticles are found to have excellent colloidal stability for days together without setting down. Capping with nontoxic l-histidine should be found to provide a hydrophilic and biocompatibility surface coating, capable of rendering good colloidal stability to ZnO nanocrystals. These biocompatible nanoparticles having average size less than the size of the protein molecules will offer prospects as promising biolabels in bioimaging application.