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11 (
6
); 897-909
doi:
10.1016/j.arabjc.2017.12.018

In-situ synthesis of CuO nanoparticles in P(NIPAM-co-AAA) microgel, structural characterization, catalytic and biological applications

, , ,
a Department of Chemistry, Tsinghua University, Beijing 100084, China
b Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan
c Department of Chemistry, University of Azad Jammu & Kashmir, Muzaffarabad 13100, Pakistan
d Department of Environmental Science & Engineering, China University of Geosciences, Wuhan, China
e International Water, Air & Soil Conservation Society (INWASCON), 59200 Kuala Lumpur, Malaysia

⁎Corresponding authors at: Department of Environmental Science & Engineering, China University of Geosciences, Wuhan, China (M.A. Ashraf). m_sidiq12@yahoo.com (Muhammad Siddiq), aqeel@cug.edu.cn (Muhammad Aqeel Ashraf)

Received: , Accepted: ,
© The Author(s) 2017
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

The production of new multi-responsive poly(N-isopropylacrylamide-allyl acetic acid) [P(NIPAM-AAA)] copolymer microgel by free radical emulsion polymerization is reported. Inside this copolymer microgel CuO nanoparticles were generated by in situ reduction of copper nitrate followed by the air oxidation process, their fabrication is confirmed by powdered X-ray diffraction analysis and the average size of CuO NPs was found to be 24 nm having monoclinic shapes. By using dynamic laser light scattering the swelling & de-swelling behaviour of the pure microgel was examined at different temperature and pH values. The copolymer microgel becomes unstable at low pH as well as at high temperature values respectively. UV–visible spectra show a red shift in surface plasmon resonance λSPR of CuO nanoparticles. The catalytic property of hybrid microgel was inspected by observing the reduction of 4-nitrophenol into 4-aminophenol in the presence of excess NaBH4 with various concentrations of catalyst at room temperature. The hybrid microgels also have good anti-bacterial activity against both Gram-positive (C. albicans) and Gram-negative (E. coli) bacteria.

Keywords

Polymerization
NIPAM-AAA microgel
CuO NPs
Catalytic activity
1

1 Introduction

The emergent infectious ailments and the development of drug resistance in the pathogenic microorganisms and fungi at a shocking level is a matter of severe apprehension. In spite of the increased information of microbial pathogenesis and uses of advanced therapeutics, the disease and mortality accompanying with the microbial infections remains high until now (Song et al., 2013; Kong et al., 2008). Consequently, there is a persuasive need to discover innovative strategies and recognize new antimicrobial agents from inorganic and natural substances to grow the next generation of medicines to control microbial toxicities. Inorganic antimicrobials such as copper and silver were used from the earliest times to treat microbial poisons, prior to the widespread use of chemotherapeutics in recent health care schemes (Vogt et al., 2007; Pantic, 2014; Cerretti et al., 2017). During the past few years, nanoscale materials have attracted extensive attention due to their unique properties. It is widely accepted that these properties are not only closely related to their sizes but also to their shapes. Therefore, controlling the morphologies of nanomaterials is one of the most important issues and effective ways to obtain desirable properties (Halim et al., 2017; De’nan et al., 2017). The preparation of metal nanostructures has received much attention because of their potential applications in the fields of information storage, catalysis, electronics, and optics. As shape-controlled synthesis was addressed, nanostructures with various regular shapes such as cubes, polyhedral, wires, prisms, and rods were fabricated using a variety of methodologies (Das and Prusty, 2012; Cioffi et al., 2005; Rout et al., 2007; Zhang et al., 2008; Xu et al., 2009; Hassan and Ismail, 2017).

In the recent times, the advances in the field of nanoscience and nanotechnology has brought to fore the nanosized inorganic and organic particles which are finding increasing applications as amendments in industrial, medicine and therapeutics, synthetic textiles and food packaging products (Luechinger et al., 2007; Rahman et al., 2017; Ismail and Hanafiah, 2017). Nanoparticles usually ranging in dimension from 1 to 100 nm (nm) have properties unique from their bulk equivalent. With the decrease in the dimensions of the materials to the atomic level, their properties change. The nanoparticles possess unique physico-chemical, optical and biological properties which can be manipulated suitably for desired applications (Sharma et al., 2011; Aziz and Hanafiah, 2017). Moreover, as the biological processes also occur at the nanoscale and due to their amenability to biological functionalization, the nanoparticles are finding important applications in the field of medicine (Gul et al., 2013; Khan et al., 2017). The nanoparticles are broadly grouped into organic and inorganic nanoparticles. The latter have gained significant importance due to their ability to withstand adverse processing conditions (Chen and Li, 2010; Aslam et al., 2017). Currently, the metallic nanoparticles are thoroughly being explored and extensively investigated as potential antimicrobials. The antimicrobial activity of the nanoparticles is known to be a function of the surface area in contact with the microorganisms. The small size and the high surface to volume ratio i.e., large surface area of the nanoparticles enhances their interaction with the microbes to carry out a broad range of probable antimicrobial activities. Metal nanoparticles with antimicrobial activity when embedded and coated on to surfaces can find immense applications in water treatment, synthetic textiles, biomedical and surgical devices, food processing and packaging (Dou et al., 2012; Roslan et al., 2017). Moreover, the composites prepared using metal nanoparticles and polymers can find better utilization due to the enhanced antimicrobial activity. Copper oxide (CuO) nanoparticles have been of great interest due to its potential applications in many important fields of science and technology such as gas sensors, magnetic phase transitions, catalysts and superconductors (Das and Prusty, 2012; Razali et al., 2017a). In the past decades, great efforts have been made to study the preparation of nanosized CuO. Conventional methods for the preparation of CuO powders include one step solid state reaction at room temperature, thermal decomposition of copper salts, mechanical milling of commercial powders, and so on (Ngai et al., 2006; Nishat et al., 2011; Rahman et al., 2013; Gul et al., 2012; Razali et al., 2017b). However, none of these methods seems to be suitable for the preparation of highly dispersed CuO nanoparticles, which has been found to be an obstacle to many applications, especially in catalysts and electrodes.

Polymeric nanocomposites embedded with inorganic nanoparticles (NPs) have attracted much interest due to their high homogeneity, flexible processability and tunable physical properties such as mechanical, magnetic, optical, electric and electronic properties (Das and Prusty, 2012; Gul et al., 2012; Medeiros et al., 2011; Liu et al., 2012; Shen et al., 2008). Furthermore, cheap ceramic nanoparticles within a polymeric matrix make the nanocomposites suitable for potential applications in electronic devices such as photovoltaic (solar) cells and magnetic data storage. Heskin in 1968 reported thermal phase transition property of poly(N-isopropylacrylamide) for the first time. Since that time this exclusive polymer has continued to gain reputation. Because of their prospective applications in the field of biomedical science, numerous responsive polymeric systems, such as those tempted by light, salt, pH, thermal, co-solvent, magnetic and electric field have been synthesized and characterized b (Das and Prusty, 2012; Shen et al., 2008; Nordin et al., 2017). Though homopolymer and their polymer microgels experience phase transitions by change in external stimuli, block copolymers, however, self-assemble into various nanostructures. Such reversible phase transitions and self-assembly behaviors have generated many robust structures that can be applied in personal/home care, coating industries, drug/protein/DNA delivery, petroleum and separation processes (Yamamoto et al., 2007).

Stimuli-responsive polymers have been extensively investigated for the development of smart materials for various applications. Diverse types of stimuli, such as temperature, pH, or light, can affect the properties and conformation of polymer chains (Rai and Bai, 2011). Thermoresponsive, water-soluble polymers exhibiting a lower critical solution temperature (LCST) in water have been increasingly investigated for nanotechnology and biotechnology applications. A variety of applications, including phase separation immunoassays, hyperthermia-induced drug delivery, and environmentally responsive Pickering emulsions, have been reported. Poly(N-isopropylacrylamide) (PNIPAM), which displays a LCST in water around 32 °C, has been the most studied Thermoresponsive polymer targeting biological applications. In addition, new Thermoresponsive water-soluble polymers were developed by the introduction of nonionizable hydrophilic moieties, such as oligo(ethylene oxide) groups, into copolymers (Das and Prusty, 2012; Ngai et al., 2006; Barbero et al., 2009; Shah et al., 2012; Behrens, 2011).

Polymer metal nanocomposites are a viable choice but very little is known about their biological properties. Here, a polymer based nanocomposite loading stabilized copper nanoparticles is proposed as a biostatic coating and systematic correlations between material properties and biological effects are established. Experimental proof of the nanocomposite capability to release metal species in a controlled manner and eventually to slow or even inhibit the growth of living organisms, such as fungi and other pathogenic microorganisms, are provided. The biostatic activity is correlated to the nanoparticle loading that controls the release of copper species, independently evaluated by means of electro-thermal atomic absorption spectroscopy. Insights into the understanding of the controlled releasing process, involving CuO dissolution through the nanoclusters stabilizing layer, are also proposed Rai and Bai, 2011. As a p-type semiconductor with a narrow band gap (1.2 eV), cupric oxide (CuO) has been widely exploited for a number of interesting properties (Song et al., 2013; Kong et al., 2008; Vogt et al., 2007; Pantic, 2014; Cerretti et al., 2017). Because of its photoconductive and photochemical properties, CuO is a promising material for fabricating solar cells and lithium ion batteries (Das and Prusty, 2012; Cioffi et al., 2005; Rout et al., 2007; Zhang et al., 2008; Xu et al., 2009; Zhu et al., 2004). Furthermore, because CuO has complex magnetic phases and forms the basis for several high-Tc superconductors and materials with giant magnetoresistance (Xu et al., 2009), it has been used in the preparation of a wide range of organic-inorganic nanostructured composites that possess unique characteristics such as high thermal and electrical conductivities as well as high mechanical strength and high temperature durability (Thomas et al., 2007). Therefore, on the basis of the fundamental and practical importance of CuO nanomaterials, well defined CuO nanostructures with various morphologies have been fabricated. Azam et al have presented that the nano-sized particles of pure ZnO, CuO, and Fe2O3 were manufactured by the sol–gel combustion scheme. XRD and TEM results exhibited that ZnO nanoparticles were smallest (18 nm) in size equated to CuO (22 nm) and Fe2O3 (26 nm). Also, the antibacterial activity of all the three manufactured NPs was compared and varied significantly. Antibacterial activity enlarged with rise in surface-to-volume ratio due to a decrease in particle size of NPs. Here ZnO NPs indicated excellent bactericidal potential despite the fact iron oxide nanoparticles had the least bactericidal activity. His results indicate that NPs were most operative against Gram-positive bacterial strains compared to Gram-negative bacterial strains (León et al., 2013; Azam et al., 2012).

In order to investigate the catalytic-activities of the synthesized CuO NPs inside the microgels reduction of p-nitro-phenol (P-NP) to p-amino-phenol (P-AP) was selected as a typical reaction. This reaction was selected due to ease of monitoring because of formation of only one product and the extent of reaction can be monitored by observing absorbance at 400 nm and 300 nm with the help of UV–visible spectrophotometer (Zhang et al., 2004). In addition nitrophenol are most of the harmful pollutants that can be contaminated in industrial wastewater. P-nitrophenol and analogous are liberated from important industrial-processes such as herbicides, pesticides, insecticides, and synthetic-dyes. This extensive occurrence of P-NP inspired to proceed this reduction reaction. Moreover P-AP is less poisonous than P-NP and there is pronounced response of P-AP in many industrial processes. Therefore this reaction is academically as well as scientifically very important (Chowdhury, 2011).

We recently addressed the in-situ synthesis of CuO NPs inside the P(NIPAM-AAA) copolymer microgels with monomers of AAA and NIPAM via conventional reduction method. Different techniques including FT-IR, dynamic laser light scattering and UV–visible spectroscopy were used to characterize hybrid microgels. The objective of the current work is not only investigating the effects of AAA content on temperature and pH-sensitivity of P(NIPAM-AAA) microgels but also to tune the catalytic and biological properties of CuO nanoparticles by varying its concentration.

2

2 Experimental section

2.1

2.1 Chemicals used

Poly-N-isopropylacrylamide P(NIPAM). Allyl Acetic Acid (AAA), N,N-methylenebisacrylamide (BIS), ammonium-persulphate (APS), sodium dodecyl sulphate, Copper nitrate Cu(NO3)2, sodium borohydride (NaBH4) and 4-nitrophenol (4-NP) were used as received. Deionized water was used during the course of the experimental work. The list of chemicals is given in the following table (see Table 1).

Table 1 Chemicals used.
Sr. no. Chemicals Abbreviation Mol. weight (g/mol) Supplier
1 N-isopropylacrylamide NIPAM 113.16 Aldrich
2 N,N methylenebisacrylamide BIS 154.17 Alpha Aesar
3 Allyl acetic acid AAA 100.12 Aldrich
4 Ammonium persulphate APS 228.20 Scharlau
5 Sodiumdodecyl sulphate SDS 288.38 Fluka
6 Copper nitrate Cu(NO3)2 187.56 Aldrich
7 4-Nitrophenol 4-NP 139.11 Aldrich
8 Sodium borohydride NaBH4 37.83 Aldrich

2.2

2.2 Synthesis of P(NIPAM-AAA) copolymer microgels

The given microgels were synthesized by free-radical copolymerization of NIPAM, AAA and BIS by using APS as an initiator. The feed composition of microgels is mentioned in Table 2. A mixture of NIPAM, AAA, BIS and SDS was added in 95 ml deionized water in a 250 ml three necked round bottom flask that is fitted out with a magnetic stirrer a condenser and a thermometer under the reflux of nitrogen gas to create inert-atmosphere which prevent the oxidation of monomers and enhance polymerization process. The stirring is continuing for 30 min & heated up to 70 °C. After 30 min 5 ml of APS (0.05 M) was added in the reaction mixture to initiate the polymerization process. The reaction was allowed to continue for six hours. The prepared P(NIPAM-AAA) copolymer microgels were purified by decantation and washed with water. The process of dialysis was used to further purify the P(NIPAM-AAA) copolymer microgels for 14 days against frequently changed water at room temperature to remove, unreacted monomers & surfactant.

Table 2 Feed composition of P(NIPAM-AAA) microgels particles.
NIPAM (mol%) AAA (mol%) BIS (mol%) SDS (g) APS (0.05 M)
84 11 5 0.05 5 ml

2.3

2.3 In-situ synthesis of CuO NPs in P(NIPAM-AAA) copolymer microgels

Hybrid microgels with CuO nanoparticles restrained inside were synthesized from P(NIPAM-AAA) microgels. 15 ml of pure microgel was diluted with deionized water up to 50 ml in a 100 ml round bottom flask. 0.2 ml (0.1 M) Cu (NO3)2solutions were added in the reacting mixture. It was stirred for one hour at room temperature under N2 reflux. After that freshly prepared NaBH4 solution (0.02 g in 5 ml water) was added drop wise to the reacting mixture. As soon as NaBH4 solution was added in the reaction mixture the growth of the NPs took place and color of reaction mixture was turned into brownish black. The reaction mixture was further stirred for one and a half hour in order to complete the growth of the nanoparticles. The resulting hybrid microgels loaded with CuO NPs was purified by decantation and 2hrs dialysis against regularly changed water. The feed composition is for the production of hybrid Microgels is also tabulated in Table 3.

Table 3 Feed compositions for P(NIPAM-AAA)-CuO hybrid microgels.
Pure microgel (ml) D.I. water (ml) Cu(NO3)2 solution (0.1 M) NaBH4 solution (1.06 M)
5 40 0.05 ml 5 ml
5 40 0.08 ml 5 ml
5 40 0.10 ml 5 ml

2.4

2.4 Catalytic activity of hybrid microgel

To study the catalytic activity of hybrid microgels reduction of 4-nitrophenol by NaBH4 was monitored as a typical reaction. The reaction procedure was such that 5.0 ml of freshly prepared 1.06 M solution of NaBH4 was added in 5.0 ml of 0.1 mmol/L solution of p-nitrophenol. The color of the solution turned from light-yellow to bright-yellow. The pH of 4-nitrophenol solution was adjusted to 10 by NaOH solution. Subsequently different amounts (0.05 ml, 0.08 ml and 0.10 ml) of highly diluted hybrid microgels were added one by one in the mixture of above solution. Suddenly after the addition of hybrid microgels UV–visible spectra of the solution were noted after specific time intervals. The rate constant of this reduction reaction was calculated by taking the UV–visible absorption spectrum of p-nitrophenol as a function of time.

2.5

2.5 Biological activities of CuO nanoparticles

Antibacterial and antifungal activities of the manufactured CuO NPs were done against both Gram-positive (C. albicans) and Gram-negative (E. coli) bacteria. The antibacterial activity was done by revised Kirby-Bauer disk diffusion, method. In short the pure cultures of organisms were subcultured in Müller-Hinton broth at 37 °C ± 3 °C on a rotary shaker at 150 rpm. For bacterial-growth a lawn of culture was arranged by spreading the 200 μL fresh culture having 106 colony forming units (CFU)/mL of every test bacterium on nutrient agar plates, with the help of a sterilized glass-rod diffuser. Dishes were left standing for 15 min to let the culture get fascinated.

At that time 10 mm wells were pressed into the nutrient agar plates for testing nanomaterial antiseptic activity. Wells were closed with one drop of melted agar to avoid leakage of nanomaterials from the bottom of the wells. Using a micropipette 200 μL of the sample of NPs suspension was poured onto each of five wells on all dishes. Subsequently overnight incubation at 37 °C ± 3 °C the different levels of region of inhibition was measured. Solvent blank was used as (−ve) control. Antibiotic tetracycline was used as a (+ve) control.

2.6

2.6 Characterization

Following different techniques were used for the characterization of microgels.

2.6.1

2.6.1 Fourier transform infrared (FTIR) spectroscopy

The FTIR spectrum of pure microgels and hybrid microgels was recorded in a dried form with Nicole’s FTIR Nexus 470 spectrometer.

2.6.2

2.6.2 Dynamic laser light scattering

Laser Light Scattering measurements were performed by means of commercial laser light scattering equipment (Brookhaven) consisting of a BI-200SM motor-driven goniometer and BI-9025AT digital autocorrelation or BI-9025AT photon counter at scattering angle 90°. A cyclindrical 22 mW uniphase He-Ne laser (637 nm) with a pinhole of 100 nm and Contin software was used.

2.6.3

2.6.3 UV–visible spectroscopy

The UV–visible 1601 Shimadzu spectrophotometer with wavelength range of 250–800 nm was used to study the optical properties of CuO NPs. Kinetics of catalytic reduction of p-nitrophenol was also studied by using this technique.

2.6.4

2.6.4 X-ray diffraction analysis (XRD)

Powdered XRD spectra were taken on Philips X’Pert Pro 3040/60 diffractometer fitted out with Cu Kα radiation source at 40 KV and 30 mA currents.

2.6.5

2.6.5 Transmission electron microscope (TEM) analysis

For transmission electron microscopy TEM model HITACHI S5500, installed in Tsinghua University, Analysis center has been used.

3

3 Results and discussion

3.1

3.1 Physical appearance of P(NIPAM-AAA) microgels

Multiresponsive P(NIPAM-AAA) microgels were synthesized by free-radical polymerization process. At 70 °C persulphate ions (S2O82−) which are produced from initiator (APS) decompose and produce sulphate radicals (SO4•) which initiate the polymerization process. After initiation the monomer of NIPAM polymerizes and the chain starts to grow once the chain length touches a certain critical length it breaks down giving precursor-particles. The precursor particles then grow by two ways either by aggregation with other precursor-particles or by absorbing in existing colloidally stable particles. The microgel gets stabilizes due to charge that is imparted by the initiator. To synthesize the microgels it is necessary to stabilize the precursor particles in early stage of reaction. Due to the absence of charge required to stabilize the small precursor particles surfactant is added. After 20 min of polymerization process the solution’s color changed from transparent to milky dispersion that indicates the microgel’s formation. This milky form is due to the change in light scattering caused by an alteration in the dimension of the microgels.

3.2

3.2 Fourier transform infrared spectroscopy

The FTIR spectroscopy was used to classify the different functionalities present in the microgels. The FTIR spectra of pure P(NIPAM-AAA) microgels and hybrid P(NIPAM-AAA)-CuO are revealed in Fig. 1 and the values are mentioned in Table 4. All the peaks have been under gone a blue shift due to the increase in the compactness and bond energy increases by the loading of CuO nanoparticles inside the copolymer microgel.

FTIR spectra of (a) P(NIPNM-AAA) microgel, (b) P(NIPNM-AAA)-CuO hybrid microgels.
Fig. 1
FTIR spectra of (a) P(NIPNM-AAA) microgel, (b) P(NIPNM-AAA)-CuO hybrid microgels.
Table 4 Observed peaks for pure P(NIPAM-co-AAA) microgel in FTIR spectra.
Chemical bond/functional group Observed peak for microgel (cm−1) Literature values (cm−1)
N—H (stret) 3272.9 3490–3250
C—H (Aliph.) 2960.2–2880 2800–3200
C⚌O (amide) 1620.6 1800–1500
N—H (bend.) 1537 1600–1500
C—N (stret.) 1367 ∼1400
C—H (bend.) 1460.9–1390 1470–1350

For C—H stretching two peaks at 2960.2 cm−1 for asymmetric and at 2880 cm−1 for symmetric stretching were observed. For amide group C⚌O a characteristic peak appeared at 1620.6 cm−1peak for N—H appeared at 1537 cm−1 for C—N peak appeared at 1367 cm−1 for —CH2— and —CH3 (bending) peaks appeared near 1460.9 and 1390 cm−1 respectively. In case of NIPAM a strong peak (at 1655.2 cm−1) was observed in the range of 1640–1680 cm−1 which is specific for —C⚌C— double bond. But there is no peak in this region for microgels.

The absence of double bond confirms the polymerization. Also in cross-linker there is a peak (at 3032.2 cm−1) just above 3000 cm−1 which is a characteristic peak for —C—H (stretch) but there is no peak just above 3000 cm−1 in case of microgels no solid peaks in the range of 610–990 cm−1 conforming the stretching mode of allyl double bonds were observed in FTIR spectra of microgels as were observed for monomers and cross linker. The broad and intense peak at 3458.6 cm−1 gives characteristic peak for N—H stretching showing the hydrogen bonding for water attached to polymer. These results indicate that polymerization occurred, and gel was formed and loaded with CuO nanoparticles (see Table 5).

Table 5 Observed peaks for P(NIPAM-co-AAA)-CuO hybrid microgel in FTIR spectra.
Functional group Observed peak for microgel (cm−1) Literature values (cm−1)
N—H (stret) 3458.6 3500–3300
C—H (Stret) 2970.0–2847.1 2800–3200
C⚌O (amide) 1736.6 1800–1500
N—H (bend.) 1532.5 1600–1500
C—N (stret.) 1365.9 ∼1400
C—H (bend.) 1454.9–1365.8 1470–1350

3.3

3.3 Temperature sensitivity of P(NIPAM-AAA) microgels at different pH values

The synthesized P (NIPAM-AAA) microgels were found to be temperature sensitive as well as pH sensitive. The multiresponsive behaviour of P(NIPAM-AAA) microgel is due to presence of P(NIPAM) component which is temperature sensitive and AAA component which is pH sensitive. The temperature sensitivity of microgel was studied at different pH values of 3, 7 and 12 in terms of change in hydrodynamic radius (Rh) values measured by LLS at scattering angle of θ = 90°. The microgel dispersions of various pH values were prepared using very dilute HCl and NaOH aqueous solutions. From dynamic laser light scattering it was observed that size of microgel increases as we increase the pH as shown in Fig. 3.

This swelling of microgel is due to deprotonation of allyl acetic acid segments present in microgel. Due to this deprotonation, carboxylate ions are produced inside the microgel and electrostatic repulsion is created between these ions which results in swelling of microgel. It is clear from the figure that the increase in hydrodynamic radius of microgel particles as we increase the pH. At low pH magnitude of the microgel particle size is lesser but as the pH increases the size of microgel particle increases as manifested by increase in hydrodynamic radius. This is due to the fact that with the increase in pH dissociation of carboxyl groups takes place and charge density increases in microgel network.

The NIPAM component in the P(NIPAM-AAA) microgel network undergoes a volume phase transition from extracted to contracted state by rise in temperature. The driving force for thermal sensitive volume phase transition was reflected as a balance between hydrophobic-hydrophilic interactions between network-chains and water molecules. The temperature at which size of the microgels changes abruptly is called volume phase transition temperature (VPTT). The entropically preferred exclusion of water from the polymer matrix along with hydrophobic and hydrogen-bonding interactions between adjacent polymer-chains allows the particles to suffer a large volume change. This transition is due to decrease in solvency of water molecules for NIPAM component in the microgels network. The amount of swelling is controlled by the free energy changes connected with the network-elasticity and mixing of the polymer & water molecules. As a result of this phase transition the state of water molecules in the gel changes from bound water to free water molecules (see Fig. 2).

Hydrodynamic radius (Rh) of P(NIPAM-AAA) copolymer microgels as afunction of temperature at various pH values.
Fig. 2
Hydrodynamic radius (Rh) of P(NIPAM-AAA) copolymer microgels as afunction of temperature at various pH values.
Hydrodynamic radiuses (Rh) of P (NIPAM-AAA) microgelsas a function ofpH at various temperatures.
Fig. 3
Hydrodynamic radiuses (Rh) of P (NIPAM-AAA) microgelsas a function ofpH at various temperatures.

3.4

3.4 pH sensitivity of P(NIPAM-AAA) copolymer microgels

The variations in the pH of P(NIPAM-AAA) copolymer microgels was studied as a function of (Rh) at 25 °C. Change in hydrodynamic radius of P(NIPAM-AAA) microgel particles vs pH is revealed in Fig. 4.3. As anticipated the functional groups AAA in the copolymer-microgels network caused in pH sensitive volume phase transitions. Microgel particles show a continuous volume change in three phases with an increase in pH. The first phase was observed at pH values below the pKa value of AAA where the size of microgels persisted almost constant. The second phase was observed when the pH was near the pKa value of AAA. In this phase of AAA groups started to deprotonate and ionized. Due to this deprotonation charge density in the copolymer network increased and electrostatic repulsion was created between ionized AAA groups (see Fig. 4).

Hydrodynamic radiuses (Rh) of P (NIPAM-AAA) microgelsas a function ofpH at various temperatures.
Fig. 4.3
Hydrodynamic radiuses (Rh) of P (NIPAM-AAA) microgelsas a function ofpH at various temperatures.
UV–visible absorption spectra of (i) CuO nanoparticles (ii) P(NIPAM-co-AAA)-CuO.
Fig. 4
UV–visible absorption spectra of (i) CuO nanoparticles (ii) P(NIPAM-co-AAA)-CuO.

The coulombic repulsions among the ionized AAA units increase the size of microgels network. At pH value greater than 7.0 all the AAA units are ionized and a maximum swelling ratio is attained. This pH sensitivity of P(NIPAM-AAA) microgels allows to study the change in interaction grade between the drug and gel-network chains to govern the drug release.

3.5

3.5 UV–visible spectroscopic study of P(NIPAM-AAA)-CuO hybrid microgels

UV–visible spectra of synthesized hybrid microgels were observed at 25 °C as revealed in Fig. 4.4. A red shift in surface plasmon-bands was observed. This red shift may be due to the assimilation of particle’s surface charge over a small surface area in such a way that the nearby medium cannot compensate the restoring force effectively. This results in increased electronic oscillations.

UV-Visible absorption spectra of (i) CuO nanoparticles (ii) P(NIPAM-co-AAA)-CuO.
Fig. 4.4
UV-Visible absorption spectra of (i) CuO nanoparticles (ii) P(NIPAM-co-AAA)-CuO.

3.6

3.6 X-ray diffraction analysis of pure and hybrid microgel of P(NIPAM-AAA) loaded with CuO nanoparticles

Fig. 5 signifies that pure microgel is amorphous in nature that’s why there is no peak appeared for this. It also indicates that for in situ synthesized CuO NPs different peaks were observed at (2θ) = 32.40°(1 1 0), 34.69°(1 1 1), 36.80°(1 1 1), 47.91°(3 0 2), 56.66°(0 2 0), 63.74°(2 0 2) and 67.72°(3 2 0) relates to different planes of CuO NPs. This confirms the formation of CuO NPs. Every CuO NPs has an interlayer spacing of 1.78861°A which was calculated by using Williamson Hall plot.

XRD peaks of (a) P(NIPAM-co-AAA) microgel, (b) P(NIPAM-co-AAA)-CuO hybrid microgel.
Fig. 5
XRD peaks of (a) P(NIPAM-co-AAA) microgel, (b) P(NIPAM-co-AAA)-CuO hybrid microgel.

3.7

3.7 Catalytic activity of P(NIPAM-AAA)-CuO hybrid microgel

In order to investigate the catalytic-activity of synthesized CuO NPs inside the microgels reduction of p-nitro-phenol (P-NP) to p-amino-phenol (P-AP) was selected as a typical reaction. The conversion of P-NP into P-AP in aqueous solution of NaBH4 is thermodynamically favorable but due to the large kinetic barrier a large potential difference between electron donor and acceptor suppresses the possibility of this reaction. Due to the large kinetic barrier reduction of P-NP does not proceed over times even in large excess of aqueous NaBH4 solution. However, when small amount of a nano catalyst is added in the reaction mixture then a significant decrease in the absorbance peak of P-NP and an arrival of a different peak at λ ∼ 300 nm is observed which indicates the conversion of P-NP into P-AP. In our studies small amounts of catalyst (hybrid microgels) were added in the mixture of P-NP and NaBH4 aqueous solutions. The reduction process was observed by determining UV–visible spectra at different time intervals. The height of characteristic peak of P-NP gradually decreased with time and a new peak was observed at λ ∼ 300 nm. The gradual decrease in characteristic peak of P-NP is due to the fact that as the reduction proceeds color of the reacting substances changes from bright-yellow to colorless. This catalytic effect is in accordance with results reported earlier. During this reduction process CuO-NPs present in the microgels catalyze the reaction by overcoming the kinetic barrier as they facilitate electron-relay from BH4−1 to P-NP (see Fig. 6).

Conversion of P-NP in aqueous solution by consuming 0.05 ml of dilute hybrid gel respectively as a catalyst at 25 °C.
Fig. 6
Conversion of P-NP in aqueous solution by consuming 0.05 ml of dilute hybrid gel respectively as a catalyst at 25 °C.

It is also important to note that both the p-nitrophenol (P-NP) and catalyst (hybrid gels) absorb at same wavelength. But during catalytic reduction of P-NP absorbance measurements of P-NP are not affected by the presence of catalyst due to very low concentration of catalyst in the reaction mixture. It should be noted that the UV–visible spectra only reveal species in solution and not the intermediates on the surface of the particles. Analogous UV–visible spectra were acquired for all systems under examination. The time delay may also be due to the reason that the catalyst is first activated and then starts its catalytic action. Activation of catalyst takes time as a result a time delay is observed in this catalytic reduction reaction.

As the concentration of NaBH4 was chosen 100 times larger as compared to that of P-NP so we assumed that the rates of these reduction reactions were independent on the concentration of NaBH4 because it was taken in large excess. Therefore, these reduction reactions can be treated as pseudo first-order reactions in phenol concentration. So, to evaluate the kinetic rate-constant pseudo first-order kinetics with respect to the P-NP concentration was used. The kinetic equation is given as follows.

(1)
dC t dt = - k app C t

By integration the above equation can be modified to the following form:

(2)
ln C t C o = - k app t where Ct and Co are the concentrations of P-NP at time t and 0 respectively and kappis apparent rate constant. Since the absorbance of P-NP is directly proportional to its concentration in the reaction mixture so the concentration ratio Ct/Co of P-NP can be calculated from the ratio of absorbance of P-NP at time t (At) to that at time 0 (Ao). Apparent rate constant was calculated by plotting ln (At/Ao) against time as shown in Fig. 4.8. With the increase in concentration of catalyst an increase in apparent rate constant at room temperature was observed as shown in Fig. 4.9. The values of apparent rate-constants were estimated from the slopes of linear portions of plots and are mentioned in the following Table 6 (see Fig. 7.).
Tem Images of (a) healthy and (b) unhealthy bacterium E. coli.
Fig. 4.8
Tem Images of (a) healthy and (b) unhealthy bacterium E. coli.
Tem Images of (a) Cells treated with 170 µg/ml of hybrid microgel (b)Cells treated with 400 µg/ml of hybrid microgel.
Fig. 4.9
Tem Images of (a) Cells treated with 170 µg/ml of hybrid microgel (b)Cells treated with 400 µg/ml of hybrid microgel.
Table 6 Antibacterial activity of CuO NPs against Escherichia coli and Candida albicans laboratory bacterial strains.
Bacterial-strains used Zone of inhibition Z.O.I. (mm) Minimum inhibitory concentration M.I.C. (μg/mL) Minimum bactericidal concentration MBC (μg/mL)
E. coli 17.2 ± 0.9 104 ± 4.7 126 ± 6.5
C. albicans 15.5 ± 0.7 122 ± 8.1 136 ± 9.8
(a) Effect of concentration of catalyst on apparent rate-constant at 25 °C. Plots of ln(Ct/Co) vs. time for the conversion of 4-NP by consuming (b) 0.05 ml (c) 0.08 ml and (d) 0.1 ml hybrid gel respectively.
Fig. 7
(a) Effect of concentration of catalyst on apparent rate-constant at 25 °C. Plots of ln(Ct/Co) vs. time for the conversion of 4-NP by consuming (b) 0.05 ml (c) 0.08 ml and (d) 0.1 ml hybrid gel respectively.

3.7.1

3.7.1 Biological activities of P(NIPAM-co-AAA) hybrid microgels loaded with CuO nanoparticles

In P (NIPAM-co-AAA) microgel CuO were fabricated by in-situ reduction of copper cations followed by air oxidation. CuO nanoparticles have been found to be biologically active and have been reported to be active against strains of bacteria and fungi.

3.7.2

3.7.2 Activity of CuO nanoparticles loaded in P(NIPAM-co-AAA) against bacterium E. coli

CuO nanoparticles are reported to be active against bacterium (E. coli). E. coli is a very common bacterial strain and normally available in molecular biology, microbiology and biochemistry laboratories. It is logical to state that binding of copper nanoparticles to bacteria depends on the surface area available for interaction. As nanoparticles have a large surface area, their bactericidal efficacy is enhanced compared to large sized particles; hence, they are believed to impart cytotoxicity to microorganisms. The mechanism by which nanoparticles are able to penetrate into bacteria is not understood completely but studies have suggested that when E. coli is treated with copper oxide nanoparticles changes take place in its cell membrane morphology. I treated the strains of standard E. coli with 5 μM solution of P(NIPAM-co-AAA) hybrid microgel loaded with CuO nanoparticles. E. coli bacterium culture treated with few drops of hybrid microgel was examined by transmission electron microscope and deaths of bacterial cells were evident as shown in following figures. Images of healthy and unhealthy E. coli before and after treating with CuO nanoparticles are shown below. The results of the antibacterial action of CuO NPs against these germs are presented in the following table.

The current investigated values showed in above table are found that CuO NPs in situ synthesized in P(NIPAM-co-AAA) microgels exhibit somewhat higher MIC values than the previously reported MIC values. Grounded on, these results, it can be decided, that the in-situ synthesized CuO NPs had substantial antibacterial-actions against the Gram positive and Gram negative bacteria. The antibacterial action of CuO NPs, against the Gram(−ve) bacteria was greater as compared to the Gram(+ve) bacteria. The variance in activity against these two types of bacterial species is because of the compositional & structural dissimilarities of the cell membranes of bacteria (see Fig. 8.).

Tem Images of (a) healthy and (b) unhealthy bacterium E. coli.
Fig. 8
Tem Images of (a) healthy and (b) unhealthy bacterium E. coli.

3.7.3

3.7.3 Activity of CuO NPs in situ synthesized in P(NIPAM-co-AAA) against Fungi Candida Albicans

Candida albicans is a very famous kind of diploid fungi possessing filamentous cells. It can cause opportunistic-oral and genital-infections in humans. C. albicans were incubated in the presence of different amounts of Nano-CuO for 24 h at 25 °C. Fungal cells were treated with 170 µg/ml and 400 µg/ml of hybrid microgel. Hybrid Microgel with CuO nanoparticles inhibits the normal budding process by penetrating inside the cells and destructing their membrane integrity. Strain of fungi C. albicans treated with hybrid microgel loaded with CuO nanoparticles are shown in following figures (see Fig. 9.).

Tem Images of (a) cells treated with 170 µg/ml of hybrid microgel, (b) cells treated with 400 µg/ml of hybrid microgel.
Fig. 9
Tem Images of (a) cells treated with 170 µg/ml of hybrid microgel, (b) cells treated with 400 µg/ml of hybrid microgel.

4

4 Conclusions

CuO nanoparticles inside a new multi-responsive P(NIPAM-co-AAA) copolymer microgel were successfully synthesized. X-ray diffraction analysis confirmed the fabrication of CuO nanoparticles. Optical properties of copper oxide nanoparticles were studied by UV–visible spectrophotometer and a red shift in surface plasmon resonance band was observed. XRD, FTIR and UV–visible results validated the similarity of manufactured CuO NPs. Results showed that at low pH and high temperature conditions aggregation takes place and microgel was found to be unstable at these conditions respectively. The copolymer hybrid microgel containing CuO NPs were evaluated for catalysis for the reduction of p-nitrophenol into p-aminophenol. The reduction rate was increased by increasing the amount of catalyst. The maximum apparent rate constant was determined to be 75.5 × 10−2 min−1 at 25 °C. Results also revealed that microgel act as an ideal system for the fabrication of metal nanoparticles and to employ the metal nanoparticles as catalyst and the in situ synthesized CuO NPs were found to be very stable. In biological studies it was found, hybrid microgels loaded with CuO nanoparticles have good biological activity against bacterium E. coli and fungi C. albicans. The antibacterial activity has been tested on Escherichia coli and C. albicans evidently validated that the smaller particle-sizes of CuO NPs have greater antibacterial properties and greater Zone of Inhibition values. The in situ method of manufacturing CuO NPs might also be prolonged to fabricate other industrially significant metal-oxides.

Acknowledgement

Special thanks to Department of Chemistry, Quaid-i-Azam University, Islamabad, National Center for Physics, Quaid-i-Azam University, Islamabad, Pakistan and School of Environmental Studies, China University of Geosciences, Wuhan, China to support this project.

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