Introduction

The naturally occurring biopolymer chitosan is made from chitin, which is a material present in the exoskeletons of crustaceans like lobsters and shrimp. It is a straight polysaccharide made up of N-acetyl-D-glucosamine and β-(1-4)-linked D-glucosamine units that are dispersed randomly [1, 2] Chitosan is notable for its unique properties, including biocompatibility [3], biodegradability [4], antimicrobial activity [5], and adsorption capacity [6, 7]. Due to its diverse range of properties, chitosan finds numerous applications in various fields [8]. In the biomedical industry, chitosan is used for wound healing [9, 10], tissue engineering [11, 12], drug delivery [13, 14] and as an antibacterial agent [5]. Its hemostatic properties make it effective in controlling bleeding in surgical procedures [15]. Chitosan’s ability to interact with metal ions and organic molecules makes it a useful material for water treatment [16]. It can be employed in the removal of heavy metal contaminants, dyes, and pollutants from industrial wastewater, as well as in filtering processes for drinking water purification [17,18,19,20].

A cyclic oligosaccharide called beta-cyclodextrin (β-CD) is obtained from natural sources like maize starch. It is made up of seven units of glucose joined together by alpha-(1, 4)-glycosidic bonds [21]. β-CD has a cone-shaped structure with a hydrophilic outer surface and a hydrophobic central cavity [22]. Due to its molecular structure, β-CD has the ability to form inclusion complexes with various hydrophobic compounds, including drugs, flavors, and fragrances [23,24,25,26]. This property makes it useful in various industries, such as pharmaceuticals [27], food [28], cosmetics [29], and agriculture [30]. In the pharmaceutical industry, β-CD is utilized as a drug delivery system to improve the solubility, stability, and bioavailability of poorly soluble drugs [31]. It can encapsulate the hydrophobic drugs within its cavity, thereby enhancing their solubility in aqueous solutions and improving their absorption in the body [32]. Overall, β-CD is an adaptable substance with a broad variety of uses in many sectors, primarily because of its capacity to create inclusion complexes and enhance the characteristics of hydrophobic substances [33].

ZnO nanoparticles (NPs), which are nano-sized particles of zinc oxide, have several applications across various fields [34]. Some of the common applications of ZnO NPs include: sunscreen [35], antibacterial agents [36], catalysis [37], gas sensing [38], optoelectronics [39], nano fertilizers [40] and wastewater treatment [41]. It is important to note that while ZnO NPs offer various advantages in these applications; their potential toxicity and environmental impact should also be considered in the development and use of such technologies [42, 43].

A unique chitosan-azo-(4-N, N-dimethylaniline) derivative was synthesized for the first time in this article as a new branch in chitosan polymers. The produced polymers were then modified using -CD and ZnO NPs to yield various polymers. These polymers are used for antifungal and antibacterial activity.

Materials and methods

Materials

Chitosan polymer and β-cyclodextrin were purchased from Merck company, Germany. Zinc oxide nanoparticles (> 97% purity, > 50 nm), glacial acetic acid (AcOH), N, N-dimethylaniline and dimethylformamide (DMF) were supplied from Aldrich, Milwaukee, Wisconsin, USA.

Synthesis of 4-nitroso-N, N-dimethylaniline

N, N-dimethylaniline (100 mlmol) dissolved in hydrochloric acid (50 ml) at room temperature in three necked conical flask. Then, add ice water till solution below 5 °C. Add drop wise of sodium nitrate (100 mlmol) in 10 ml dist. water to the last mixture. Stand the reaction mixture for one hour and then, filter it and washing the final precipitate by dil. HCl and ethanol. Stirring at room temperature the generated salt in 100 ml dist. water for 30 min and then, add solution of potassium hydroxide (10%) till the alkaline solution. Using separated funnel to collect the nitroso compound, and concentrated it to half amount. Cooling the final solution, and filtrated the precipitate by electrical pump, Scheme 1.

Scheme 1
scheme 1

Synthesis of 4-nitroso-N, N-dimethylaniline

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Synthesis of Chs-DAN polymer

Chitosan dissolved (1.0 gm) in 49 ml of distilled water and 1 ml of glacial acetic acid at room temperature. Add drop wise of the solution of 4-nitroso-N,N-dimethylaniline (0.5 gm in 10 ml methanol) at room temperature for two hours, Scheme 2. The Chs-DAN polymer was obtained as a green powder by pouring the reaction mixture into a Petri plate and letting the solvent evaporate at room temperature.

Scheme 2
scheme 2

Synthesis of Chs-DAN polymer

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Synthesis of pseudopolyrotaxane (Chs-DAN/β-CD)

Chs-DAN polymer (1.0 g) and β-CD (2.0 g) were stirred at room temperature in 50 ml of methanol for 18 h, Scheme 3. After pouring the hydrogel into a Petri plate and letting the solvent evaporate at room temperature, a pale-green powder known as Chs-DAN/β-CD was obtained.

Scheme 3
scheme 3

Synthesis of Chs-DAN/β-CD polymer

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Synthesis of Chs-DAN/ZnO NPs composite

Chs-DAN polymer (1.0 g) and ZnO NPs (0.10 g was stirred at room temperature in MeOH (50 ml) for 24 h, Scheme 4. The afforded hydrogel was poured into a Petri plate and then, dried at room temperature to afford Chs-DAN/ ZnO NPs as light green powder. Physical and chemical properties of the synthesized polymers were showed in Table 1.

Scheme 4
scheme 4

Synthesis of Chs-DAN/ ZnO NPs composite

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Table 1 Some physical and chemical properties of the synthesized polymers
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Characterizations

Fourier-transformation infrared (FTIR)

Function groups of the synthesized polymers were identified by infrared spectrometer (Jasco Model 4100 – Japan) in the range of 4000–400 cm1 wavenumber region.

X-ray diffraction (XRD)

X-ray diffraction (XRD) measurements were performed at room temperature using a powder diffract meter (Brucker D8 Advance, Germany) fitted with a Cu K radiation source, = 1.5406 and 2 in the range (5–80°), to ascertain the crystallite size and phase structure of the produced polymers.

Scanning electron microscope (SEM)

A 20 kV accelerated voltage SEM (JEOL SEM model JSM-5500 – Japan) was utilized to examine the morphological structures of the generated derivatives.

UV–visible spectroscopy

The optical characteristics of the synthesized polymers were studied by UV–visible spectroscopy (PG Instruments, model T80, UK) and quartz cells with a path length of 1 cm with wavelengths ranging from 200 to 800 nm. To modify the baseline, chloroform was utilized as a blank.

In vitro antimicrobial activity

The antimicrobial activity of pure and synthesized chitosan derivatives was examined by the diffusion method in a gar cup [44] Escherichia coli ATCC 25933 (G− ve) and Staphylococcus aureus ATCC 6538-P (G + ve), Candida albicans ATCC 10231(yeast) and Aspergillus niger NRRL-A326 (fungus) were the four reference test microorganisms used. In the case of bacteria and yeast, nutrient agar plates were severely injected on a daily basis with 0.1 ml of 105–106 cells/ml. The antifungal properties were evaluated using potato dextrose agar plates inoculated with 0.1 ml (106 cells/ml) fungal inoculum. Samples dissolved in DMSO were placed in initiated holes in inoculated plates. To enable maximum diffusion, plates were then maintained at a low temperature (4 °C) for two to four hours. After that, the plates were allowed to incubate for 24 h at 37 °C for bacteria and 48 h at 30 °C while horizontal to promote the organisms’ maximal reproduction. By measuring the diameter of the zone of inhibition and expressing the result in millimeters (mm), the antibacterial activity of the test agent was evaluated. The experiment was run multiple times, and the average reading was noted.

Results and discussion

XRD analysis

Crystallite size and its phase structure of chitosan polymer derivatives detected by XRD analysis (Fig. 1). At 25 °C, XRD spectra in the 5°–80° range were observed. Chitosan’s typical diffraction peaks were identified at 2 = 8.6° and 20°, confirming its semi-crystalline structure [45]. XRD examination revealed the synthesis of Chs-DAN (azo-chitosan polymer) by amino group nature change. Furthermore, the azo-polymer and chitosan vary in crystal size and crystallinity. The XRD of Chs-DAN revealed a small peak at 18.5°, but the chitosan peaks at 16.4° and 33.6° vanished in the Chs-DAN polymer. The synthesis of azo-polymer and breaking of hydrogen bonds reduced the crystallinity of Chs-DAN (50.6%). The XRD pattern of pseudopolyrotaxane polymer (Chs-DAN/βCD) revealed a sharp diffraction angle of 16.2° and showed a crystallinity value at 46.1%. Additionally, the crystal size in Chs-DAN and Chs-DAN/βCD was 2.8 nm and 3.4 nm, respectively. After addition of ZnO, NPs to the Chs-DAN polymer gave a different peaks in XRD analysis. A broad peak in the generated chitosan polymer became a broader peak with less intensity and some different sharp peaks showed at 13°, 19°, 22° and 28.9°. Moreover, the crystal size of Chs-DAN/ZnO NPs (4.1 nm) increased on the cost of crystallinity value (45.6%).

Fig. 1
figure 1

XRD of Chs-DAN, Chs-DAN/ β-CD, and Chs-DAN/ZnO NPs

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Fourier-transform infrared spectroscopy (FTIR)

The creation of our components was confirmed using FTIR spectra. Figure 2 depicted the distinction between Chitosan (Chs), Chs-DAN polymer as an azo-polymer, 4-nitroso-N, N-dimethyl aniline. In chitosan, –OH group characteristic peak showed at 3340 cm−1, overlaid on N–H stretching band, while the hydrocarbon bond C–H showed at 2898 cm−1. And the peaks of –NH2 and –C=O were appeared at 1600 cm−1 and 1659 cm−1, respectively. The Schiff base polymer spectra revealed –N=N– band at 1600 cm−1, confirming the synthesis of Chs-DAN polymers. Furthermore, the bending and stretching vibration peaks of the aromatic ring’s C–C bonds, as well as the stretching vibration peak of nitrosobenzene’s –C–N–, showed at 1490, 820, and 1250 cm−1, respectively [46]. The distinctive band of nitrosobenzene in the area 1500 cm-1 did not emerge in the azo-polymer spectra for clearing, suggesting that no detectable residual of free nitrosobenzene exists. Table 2 summarizes the differences in absorbance bands of Chs. and Chs-DAN polymer.

Fig. 2
figure 2

FTIR of pure Chs, DAN and Chs-DAN polymer

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Table 2 FTIR changes of pure Chs and Chs-DAN polymer
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We discovered that the hydroxyl group’s absorption band is somewhat higher and less intense than the βCD (Fig. 3). In addition, v [OH] symmetric stretching was moved to a higher frequency and CH-aliphatic peak was shifted to a lower frequency compared to βCD. Furthermore, the bending vibrations [CH2–O] and [C–O–C] were moved to lower frequencies at 1040 and 1011 cm−1, respectively. These findings supported the synthesis of pseudopolyrotaxane polymer via the interaction of Chs-DAN polymer with βCD. This result may be interpreted as follows: inserting the Chs-DAN chain into the electron-rich cavity of the cyclodextrin rings causes the frequencies to increase [47]. Table 3 shows the differences in absorbance bands of pure βCD, Chs-DAN polymer, and Chs-DAN/ βCD polymer.

Fig. 3
figure 3

FTIR of β-CD, Chs-DAN polymer, and Chs-DAN/ βCD polymer

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Table 3 FTIR changes of β-CD, Chs-DAN polymer, and Chs-DAN/ βCD polymer
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The FTIR spectra of the Chs-DAN/ZnO NPs polymer in Fig. 4 show a large absorption band at 3422 cm−1 for –OH group stretching vibrations. The absorption band at 2910 cm−1 is due to symmetric stretching of aliphatic C-H of Chs in polymer blend [44], which is considerably shifted and increased in intensity (2870 cm−1) after ZnO NPs are doped. At 1700 cm−1, the absorption band is ascribed to the C=O stretching vibration [44]. The absorption bands at 1550 and 1480 cm−1, which correspond to the polymer (N=N) group bending and the stretching vibration of (CH2–OH) group, respectively. Table 4 summarizes the differences in absorption bands of Chs-DANpolymer and Chs-DAN polymer doped with ZnO NPs.

Fig. 4
figure 4

FTIR of Chs-DAN polymer and Chs-DAN/ ZnO NPs composite

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Table 4 FTIR changes of Chs-DAN polymer, Chs-DAN/ ZnO NPs composite and ZnO NPs
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SEM analysis

Using SEM analysis for study the surface morphology of the generated polymers (Fig. 5 a–c). These images show apparent differences between the generated polymers, and the surface appearances were changed upon reaction as compared to fibrous nature of chitosan’s surface. The Chs-DAN polymer showed a rough surface with some cracks (Fig. 5a). Pseudopolyrotaxane polymer (Chs-DAN/βCD) was appeared as a slightly rough and scaly structure with smaller random particle crystal size, this image like the coral reefs. On the other hand, Chs-DAN/ZnO NPs composite displayed a heterogeneous and non-smooth surface with straps and shrinkage. From these results, it was shown that new polymers in different shapes were formed.

Fig. 5
figure 5

SEM images of a Chs-DAN, b Chs-DAN/βCD, c Chs-DAN/ZnO

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Optical properties

At 25 °C, the Chs-DAN polymer, Chs-DAN/ZnO NPs composite, and Chs-DAN/β-CD experienced UV–visible spectroscopy, with measurements obtained at 200 and 800 nm (Fig. 6). The UV–visible spectrum of Chs-DAN polymer showed a sharp peak at 365–450 nm due to the presence of double bond (–N=N–). Interestingly, reaction of Chs-DAN polymer with β-CD and ZnO NPs showed slightly shifted peak form 450 nm to 465 and 470 with high intensity, respectively. The all data approved the formation of Chs-DAN/ZnO NPs composite and Chs-DAN/ β-CD. In addition, Tauc’s formula was used to determine the energy gap of the synthesized polymers based on their UV–visible absorption spectra. For the Chs-DAN polymer, Chs-DAN/β-CD, and Chs-DAN/ZnO NPs composite, the corresponding values of Eg were 3.8, 3.6, and 3.5 eV.

Fig. 6
figure 6

UV spectra of Chs-DAN, Chs-DAN/ β-CD, and Chs-DAN/ZnO NPs

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Antimicrobial activities measurements

The results obtained in (Fig. 7) and (Table 5) examined the antimicrobial actions of starting materials and the generated polymers. Using four types of microbes G + ve (S. aureus), G − ve (E. coli), yeast (C. albicans) and fungus (A. niger). It is clear that the pure samples exhibit no effect against the used microbes, while the chitosan derivatives samples and nitroso compound exhibit a remarkable antimicrobial activity against different types of used microbes. It has been found that Chs derivatives such as Chs-DAN and Chs-DAN/βCD exhibit some antibacterial action against microbiological organisms. This effect may be attributed to increasing in the free amino acid amount, resulting in excess positive charges (W. Ding et al.) [48]. Furthermore, the Chs derivatives antimicrobial activity depending on the fact that the positive charges of Chs absorbed by the negatively charged bacterial cell surfaces resulting in the leakage of intracellular components such as proteins, resulting in cell death (Gonil et al., 2011) [49]. The Chs-DAN/βCD polymer were shown to has a small increases in the activity against microbial activity than Chs-DAN polymer (Table 5), which is probably due to the increased water solubility of Chs-DAN/βCD polymer after complexion with βCD. The increasing of water solubility may led to increased contact between pathogens and the polymer contents, then improving the antimicrobial activity of Chs-DAN polymer [50]

Fig. 7
figure 7

The antimicrobial activity for different samples studied by the cup diffusion agar method

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Table 5 The antimicrobial activity of the starting materials and the prepared polymers for different types of microbes
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The inhibition zone recorded in Table 5 shows that pure ZnO and Chs-DAN/ZnO samples show a considerable higher antibacterial activity against harmful microbes E. coli and C. albicans, as well as S. aureus and A. niger. The main effect of these synthesized polymers against pathogenic microbes arises from the release of ZnO ions from the prepared samples and diffuse through the bacterial cell membranes and suppress the enzymatic activity after interference with the nucleic acid and proteins functional groups. Consequently, the change in microbe cell structure will eventually lead to microbial suppression. Another possible mode of action for the prepared samples is the release of the active form of oxygen that initiates electrostatic interaction and alters the pathways of enzyme or DNA and moreover the bacterial cell wall [51, 52].

Determination of Minimum Inhibitory Concentration (MIC)

To confirm the activity of the prepared derivatives (ZnO, Chs-DAN/ZnO, Chs-DAN and Chs-DAN/βCD) against pathogens, Minimum Inhibitory Concentration (MIC) by broth dilution method was used against G + ve (S. aureus), G − ve (E. coli), yeast (C. albicans) and fungus (A. niger) microbes [53, 54]. The MIC values for chitosan derivatives samples are listed in Table 6. All pervious derivative samples showed different antibacterial activity with MIC values ranged from 42 to 112 mg/ml depending on tested species. ZnO and Chs-DAN/ZnO samples with higher antimicrobial activities showing MIC equal 56 and 77 mg/ml against S. aureus, 69 and 80 mg/ml against E. coli, 43 and 70 mg/ml against C. albicans and 42 mg/ml against A. niger respectively. Samples Chs-DAN and Chs-DAN/βCD exhibited decreased antibacterial activity (Table 6) showing MIC equal 105 and 86mg/ml against S. aureus, 112 and 97 mg/ml against E. coli, 78 and 72 mg/ml against C. albicans,53 and 44 mg/ml against A. niger respectively.

Table 6 Minimum inhibitory concentration (MIC) of the starting materials and the prepared polymers for different types of microbes
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Conclusion

Chs-DAN polymer was produced by stirring chitosan polymer with N, N-dimethyl nitroso aniline at room temperature. Chs-DAN was obtained and added to β-CD to generate pseudopolyrotaxane inclusion complex. ZnO NPs were then doped to make the matching composite in an ecologically friendly, affordable, and appropriate manner. FTIR and XRD tests verified these derivatives, while SEM investigation revealed notable morphological differences between them. The produced materials’ improved optical characteristics and energy band gap values were validated by UV–visible measurements. The antimicrobial activity study revealed that azo-chitosan derivatives polymers are very promising as it showed highest activities against different types of pathogenic microbes than pristine polymers.