Microwave assisted ultrafast immobilization of CuO nanostructures in paper matrices for antimicrobial applications

CuO nanoflakes with dimension of 50–150 nm × 200 nm were successfully immobilized on the surface of cellulose fibers by an ultrafast (5 min reaction time), scalable microwave assisted method. The high retention of CuO up to 70% has been phenomenal considering that the immobilization was carried out in the absence of any linker, binder or retention aid. Retention of almost 87% of the immobilized CuO nanostructures even after five consecutive recyclability steps has been unprecedented. The antimicrobial activities of the paper matrices have been investigated by deactivating both G. trabeum and E. coli. The CuO immobilized paper matrices have desisted the growth of G. trabeum up to 28 days and completely deactivated E. coli (CFU in order of 107) in the presence of visible light with exposure time of 2 h only.


Introduction
Paper, a natural biopolymer has got a wide range of applications since its journey through the history (Matsubara et al. 1995). In the last few decades, the research in paper technology was mostly devoted to the development of paper matrices for high-end applications due to the demand in different avenues. Moreover, the demand for reduction in the pollution during the manufacturing owing to some of the restrictions imposed by the environmental agencies increases further the cost of the raw materials (Matsubara et al. 1995;Gimenez et al. 2011;Ornatska et al. 2011;Mostafa et al. 2019;Alrammouz et al. 2019). Due to its biodegradability nature, the paper matrices stand tall among many advanced engineering materials (Moon et al. 2011). In fact, the raw material, cellulose, is a biopolymer with a maximum natural abundance (Moon et al. 2011). For improving various paper properties, since early 1960s, paper industries started using metal oxides, clays, mica and different ceramics powders as fillers in the paper manufacturing (Chauhan and Bhardwaj 2012;Patel 2007). This journey continued in the same fashion for the next few decades until the evolution of the nanotechnology, which renewed the interest among the researchers to find new ways to incorporate nanostructures of these metal oxides and other fillers into the paper matrices with controlled microstructures so that new and renewed properties and applications can be realized (Ogihara et al. 2012;Monk et al. 2001;Manekkathodi et al. 2010;Zeng et al. 2015;Fujiwara et al. 2017;Adel et al. 2016).
Among different approaches adopted for the incorporation of various metal oxides and hydroxides nanostructures by various research groups (Mostafa et al. 2020;Dong et al. 2018;Citak et al. 2019), the wet chemical approaches employing linkers, binders and retention aids have dominated this research area (Iguchi et al. 2003;Khatri et al. 2014). The added cost owing to the addition of these molecules and materials along with their negative impact on the application of the obtained paper matrices have further encouraged the researchers to find alternative approaches to incorporate the nanostructures where none of the linker, binder and retention aid is used (Chauhan et al. 2015a). In this direction, our research group invented a novel hydrothermal approach, where neither of these linker, binder and retention aid was used to immobilize ZnO, TiO 2 , Ce 2 O 3 , Bi 2 O 3 and Fe 2 O 3 nanostructures on the surface of cellulose fibers of the paper matrices (Chauhan and Mohanty 2014;Chauhan and Mohanty 2015;Chauhan et al. 2015aChauhan et al. , b, 2019Aggrawal et al. 2015Aggrawal et al. , 2018Aggrawal et al. , 2019. Although, the immobilization of the nanostructures on the surface of the cellulose fibers is adequate and uniformly distributed but the long experimental time between few hours to a couple of days is a major bottleneck for its usability in the industrial scale. Hence, it is important to find appropriate approach where a fast immobilization can be possible. Due to a drastic growth of pathogenic microbes and inefficacy of the common antibiotics treatment against the recently emerged superbugs (antibiotic resistant bacterial species) (Wright 2000;Willyard 2017;Spellberg et al. 2008), there is an utmost need for the development of safe and reliable antimicrobial systems. Almost 80% of the bacterial infections are caused by Gram negative bacterial species (Mahmoodi et al. 2018). The water borne diseases are mostly caused due to the unavailability of safe drinking water to the major population of the world.
The CuO is a p-type semiconductor with a narrow band-gap of 1.3-1.8 eV (Maheswari et al. 2018). Hence, the CuO nanostructures have been applied in sensing, semiconductor devices, supercapacitor, photocatalysis, catalysis and antimicrobial applications. Moreover, it has also good chemical stability (Mahmoodi et al. 2018). The CuO nanostructures were proven to be excellent antimicrobial materials because of its bio-compatibility, non-toxicity against mammals, easy and economical preparation (Maheswari et al. 2018). The nanostructures need to be in contact with bacterial or fungal cells in order to provide antimicrobial properties. But nanoparticles tend to aggregate easily hence decrease the active surfaces. To achieve great applicability, they are incorporated or immobilized on various substrates. Further, the separation of the nanomaterials for their recyclability is difficult which can be easily sorted out when these are immobilized on substrates.
In this research, a novel microwave assisted ultrafast (only 5 min experimental time) immobilization methodology has been developed, wherein the CuO nanoflakes are immobilized without the assistance of linker, binder and retention aid. The antimicrobial activity of the paper matrices has been investigated against E. coli and G. trabeum in the presence of visible light. The CuO immobilized paper matrices have been also recycled for multiple preparation steps.

Synthesis
Copper oxide (CuO) nanoflakes with varied concentrations (5-30%) have been immobilized on the cellulose fibers of the paper matrices using a single step, facile, ultrafast (5 min only), microwave

Fig. 1
Schematic representation for the ultrafast immobilization CuO nanoflakes on the paper matrices using microwave assisted method assisted synthesis method (Fig. 1). Typically, 1.6 g of softwood pulp was dispersed in 40 mL of deionized water. To it, 50 mL of xM of cupric sulphate solution already prepared in DI water was added and stirred at RT for 15 min, followed by dropwise addition of 40 mL of yM NaOH solution under continuous stirring at RT for 15 min. The values of x and y are taken based on the final concentration of the CuO in the paper matrices. The amount of the precursors and other reagents used are summarised in details in Table S2.
The whole reaction mixture has been transferred to the 250 mL round bottom flask and employed to microwave treatment in an open vessel system for 5 min at 50 °C and 300 W. After the experiment was completed, the specimens have been washed repeatedly with ethanol and DI water. It was then dried at RT. The paper sheets were fabricated by Handsheet making method using British sheet former following TAPPI test method 205 sp-02. Several other CuO precursors have also been tested for immobilization of CuO nanostructures in the paper matrices (ESI for detailed synthesis).

Characterization
Standard combustion test and thermogravimetric analysis (TGA) have been used to check the retention of immobilized CuO nanostructures in the paper matrices. In the combustion test, a known weight of the paper matrices has been burnt in the muffle furnace at 520 °C for 5 h in air. It leads to complete oxidation of the organic matter i.e., cellulose, leaving behind only inorganic copper oxide. For comparison, a blank paper sheet (without CuO) was also burnt under identical condition. The TGA analysis was carried out by heating the specimens in EXSTAR TG/ DTA6300 with a heating rate of 5 °C per min in an argon atmosphere. The thermograms of all the specimens including the blank paper sheet have been compared to estimate the CuO content. The microstructure of the blank paper and CuO immobilized paper have been carried out using the Gemini 500 FESEM (Zeiss) FESEM. Standard gold sputtering of the specimen has been performed prior to the FESEM investigation. The crystal structure of the specimens has been studied using XRD. In the present research, the XRD patterns have been recorded on Rigaku Ultima IV with CuKα radiation (λ = 1.5405 Å) at a scanning speed of 4°/min in the range of 10 to 70° of the 2θ scale. The obtained diffraction patterns were compared with ICDD (International Centre for Diffraction Data) files using Xpert high score. The oxidation state and chemical environment of the different elements present in the specimens have been investigated using the XPS. It was done on PHI-5000 VersaProbe III, ULVAC-PHI INC. The peaks were referenced at 284.8 eV, the standard binding energy of C1s. Monochromatic AlKα have been used as the X-ray source for recording the XPS datas.

Antimicrobial activity
The paper matrices were subjected to studying the antimicrobial activity, i.e., antibacterial and antifungal behaviour. The antibacterial activity has been carried out using blank paper and CuO immobilized paper matrices on E. coli, a Gram-negative bacterium in the presence of visible light for 2 h. Experiments were carried out by colony count method in accordance with standard antibacterial protocol ISO 20743 with minor modifications (Owen and Laird 2021;Liu et al. 2017;Gadkari et al. 2019). The experiments were repeated in triplicate to remove any ambiguity and the results have been interpreted in percent reduction and log reduction values in E. coli (Moghayedi et al. 2017). Typically, specimens were cut in the size of 8 cm × 1 cm and were steam sterilized in an autoclave. These paper pieces were then put into 10 mL of E. coli suspension and incubated at 37 °C for 2 h in the presence of visible light in an incubator. After that, serial dilutions of the E. coli suspension were prepared and plated on the LB plates which were incubated for 24 h at 37 °C in inverted position. The E. coli colonies were counted and CFU was calculated. Identical experiments were repeated three times in order to estimate the standard deviations in log reduction for studying the reproducibility of the results. The antifungal activity has been performed against G. trabeum, a cellulose eating fungus, which produce cellulase enzyme to destroy the paper matrices. Typically, already grown fungal colonies were picked up using bacterial loop and transferred to sterilized potato dextrose agar plates. Paper specimens were cut in the size of 2 cm × 2 cm and steam sterilized in an autoclave at 15 psig pressure for 20 min. These were then placed on the fungal colonies in the petriplates and incubated at 28 °C up to 28 days. A blue sheet was placed below the petriplates to have a better contrast so that good quality images can be taken. This will provide a clear idea about the outcome. Photographs were taken at regular intervals to check the destruction of the paper matrices by G. trabeum and investigate the antifungal activity of the prepared CuO immobilized paper matrices.

Results and discussion
The retention of CuO nanoflakes in the paper matrices have been estimated using combustion test and TGA analysis (Chauhan and Mohanty 2014; Chauhan and Mohanty 2015; Chauhan et al. 2015aChauhan et al. , b, 2019Aggrawal et al. 2015Aggrawal et al. , 2018Aggrawal et al. , 2019. The paper matrices burnt in air at 520 °C for 4 h in a muffle furnace could burn out the organic mass completely leaving behind the inorganic CuO nanostructures. The extent of immobilization can be easily investigated when the leftover mass after burning of the blank paper is compared with that of the immobilized paper matrices. The leftover mass of different paper matrices has been summarized in Table S3. To have an unambiguity, The inset shows high magnification image respectively the retention of CuO in paper matrices has also been estimated using TGA experiments. While carrying out TGA in argon atmosphere (Fig. 2), it has been observed that around 12% mass is remaining in the blank paper after burning at 800 °C, this is due to the carbonization of the organic mass as the TGA has been carried out in the argon atmosphere. The mass loss at low temperature is due to adsorbed moisture in the paper sheets (Habibie et al. 2016). To calculate the retention of CuO using TGA, the leftover mass of blank paper burnt has been deducted from the leftover mass of CuO immobilized paper matrices and is summarized in Table S3. The results of combustion test corroborate with the results of TGA analysis with a minor deviation. The DTG thermograms are given in Fig. S1 for comparison.
As the immobilization could be completed with a short reaction time of 5 min only, there was a concern on unshackle of the immobilized nanostructures from the paper matrices. In order to address this concern as well as to test the efficacy of the recyclability of the paper matrices for multiple paper making cycles, the CuO immobilized paper matrices have been redispersed in water and followed the same steps of paper making for five consecutive cycles. It was phenomenal to see that there was substantial retention of the CuO nanostructures during this recycling processes. A maximum of only 13% loss of the nanostructures was estimated even after five successive cycles as shown in Fig. 3. Moreover, the leaching of CuO has been estimated after sonicating the specimen for 20 min in water. The water specimen has been subjected to ICP-MS and only 0.1% leaching of immobilized CuO content is observed. The worth of immobilization can be realized by the reported fact that by conventional filler loading, paper lose almost 100% fillers just after three cycles whereas in case of in-situ filler loading paper matrices retain only 30% of fillers after six consecutive cycles (Ciabanu and Bobu 2009).
The microstructure of the specimens has been studied by FESEM as shown in Fig. 4. The clean surface of the cellulose fibers has been observed in blank paper matrices as shown in Fig. 4a and inset. The uniform distribution of CuO nanoflakes have been observed in the high magnification images of specimen 5CuOPa-30CuOPa (Fig. 4b-f). The increase in content of CuO is clearly visible even from the FESEM images. In the specimen 30CuOPa, the surface of the cellulose fibers is almost covered with the CuO nanoflakes. With increase in content of CuO in the paper matrices, it has been observed that, clear nanoflakes have been immobilized on the paper matrices. The breadth of the nanoflakes vary in the range from 50-150 nm whereas length extend up to 200 nm.
The phase analysis of the paper matrices has been carried out using XRD (Fig. 5). Three broad peaks below 25° in the 2θ scale with their d-values centered at 0.6041, 0.5383 and 0.325 nm can be observed. These are indexed to the (1-10), (110) and (200) reflections of cellulose-Iβ, respectively (Nishiyama et al. 2002;French 2014  The oxidation state and chemical environment of elements present in the specimens has been confirmed using XPS analysis as shown in Fig. 6. The survey scan of all the specimens is shown in Fig. 6a. In all the specimens, peaks for carbon and oxygen are present at 286.6 and 532.9 eV, respectively, coming from the cellulose matrices (Aggrawal et al. , 2015Ciolacu et al. 2010;Small and Johnston 2009). In the survey scan of CuO immobilized specimen, several peaks have been observed in the region of 930-970 eV which are originated from Cu and satellite peaks confirming the presence of Cu in the specimens (Cao et al. 2019;Tan et al. 2020). To confirm the chemical environment and oxidation state, high resolution C1s, O1s and Cu2p have been recorded. The high resolution C1s spectra as shown in Fig. 6b reveal the presence of three chemically different carbons in the structure of cellulose. The three peaks at 284.8, 286.5 and 287.7 eV could be attributed to chemically different carbon, one with ether link, second carbon linked to hydroxyl groups and third carbon having two ether links, respectively Johansson et al. 1999). The high resolution O1s spectra (Fig. 6c) reveal the presence of two chemically different oxygen attributed to hydroxyl and glycosidic oxygen present in the structure of cellulose at 531.3 and 532 eV. The third oxygen present in very less amount may be attributed to some of the end hydroxyl groups or from the tape used for the XPS sampling. The high resolution Cu2p spectra is shown in Fig. 6d. These confirm the presence of Cu in 2 + oxidation state as the XPS peaks observed at 935.5 and 954.2 eV for Cu 2p3/2 and Cu 2p1/2, respectively along with the satellite peaks at 943.3 and 962.7 eV matches well with the reported values in literature (Cao et al. 2019;Pauly et al. 2014;Sun et al. 2020). Hence the immobilization of CuO in the paper matrices have been confirmed by XPS analysis.
The antimicrobial activity of the synthesized CuO immobilized paper matrices have been carried out by deactivating both fungus and Gram-negative bacterium. The antibacterial activity has been studied using colony count method and summarized in Table S4. It has been observed that 30CuOPa specimen has deactivated the complete E. coli count of 2.8 × 10 7 within 2 h of visible light exposure. In the blank paper (specimen Pa), almost no reduction in bacterial count was observed, whereas, 40, 50, 95 and 99.99% reduction in E. coli count has been estimated in the specimens 5CuOPa, 10CuOPa, 15CuOPa and 20CuOPa, respectively. With an increase in the content of CuO in the paper matrices, there is an improvement of the antibacterial activity. The antibacterial activity has been induced by the interaction of CuO nanoflakes with the cell membrane of the E. coli which resulted in the inhibition of active cell transport process, thus induces the cell lysis (Sharmila et al. 2016;Sirelkhatim et al. 2015;Villanueva et al. 2016 Fig. 7 Reduction in E. coli growth after 2 h of visible light exposure with standard deviation get attached to the negatively charged bacterial membrane and initiate cell lysis (Taran and Rad, 2016). The permeability of the cell membrane changes after interaction with Cu 2+ (Xie et al. 2020). It also leads to damage of biochemical metabolisms of the bacterial cell. Another possibility is the generation of ROS species which further deactivates bacterial cells (Maheswari et al., 2018). Further, the experiment has been carried out in triplicate and standard deviation has been calculated in the log reduction values of bacterial colonies and given in Fig. 7. Almost similar results have been observed while carrying out the experiment for multiple times. Furthermore, antifungal activity has been studied using zone of inhibition method and results has been summarized in Table 1. The antifungal activity has been carried out for 28 days and it has been observed that specimen 30CuOPa remain intact even after 28 days of fungal infection (Table 1). The brown colour of the paper is intact in case of specimen 20CuOPa until 15 days of infection. Whereas, specimen Pa and 5CuOPa has been completely damaged by the fungal cells. The cellular wall of the fungus is composed of polysaccharides and glycoproteins. The CuO nanoflakes may get attached to the wall of the fungus which interrupts its diffusibility and damage the cell membrane. Moreover, it could also be attributed to the generation of ROS derived disruption of cellular homeostasis and dynamic equilibrium. It leads to leakage of cell contents resulting in death of fungal cell (Maheswari et al. 2018). Hence, the increase in content of CuO in paper matrices has resulted in the increase in antifungal activity as that of antibacterial activity. The material has shown promising results against both bacterial and fungal cells.

Conclusion
In summary, an ultrafast (5 min only) microwave assisted methodology has been developed to immobilize CuO nanoflakes on the surface of the cellulose fibers of paper matrices. Neither linker, binder nor retention aid has been used for the immobilization. The CuO nanoflakes immobilized paper matrices could survive five consecutive recyclability cycles without much loss of the nanostructures. All these specimens have shown good antimicrobial activity by deactivating a Gram-negative bacterium, E. coli and paper eating fungus, G. trabeum. Among all these specimens, 30CuOPa has shown best antimicrobial activity against both bacteria and fungus. The concept of ultrafast immobilization of CuO nanoflakes on the paper matrices and phenomenal antimicrobial activity has not only academic values but could also open up industrial avenues in medical and water purification fields.