IET Nanobiotechnology
Volume 14, Issue 1, February 2020
Volumes & issues:
Volume 14, Issue 1
February 2020
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- Author(s): Sania Naz ; Ayesha Gul ; Muhammad Zia
- Source: IET Nanobiotechnology, Volume 14, Issue 1, p. 1 –13
- DOI: 10.1049/iet-nbt.2019.0176
- Type: Article
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Copper oxide nanoparticles (CuO NPs) use has exponentially increased in various applications (such as industrial catalyst, gas sensors, electronic materials, biomedicines, environmental remediation) due to their flexible properties, i.e. large surface area to volume ratio. These broad applications, however, have increased human exposure and thus the potential risk related to their short- and long-term toxicity. Their release in environment has drawn considerable attention which has become an eminent area of research and development. To understand the toxicological impact of CuO NPs, this review summarises the in-vitro and in-vivo toxicity of CuO NPs subjected to species (bacterial, algae, fish, rats, human cell lines) used for toxicological hazard assessment. The key factors that influence the toxicity of CuO NPs such as particle shape, size, surface functionalisation, time–dose interaction and animal and cell models are elaborated. The literature evidences that the CuO NPs exposure to the living systems results in reactive oxygen species generation, oxidative stress, inflammation, cytotoxicity, genotoxicity and immunotoxicity. However, the physio-chemical characteristics of CuO NPs, concentration, mode of exposure, animal model and assessment characteristics are the main perspectives that define toxicology of CuO NPs.
Toxicity of copper oxide nanoparticles: a review study
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- Author(s): Kaliyaperumal Viswanathan ; Babu Diviya Bharathi ; Chitra Karuppannan ; Thiyagarajan Sanjeevi ; Madan Nithiyanantham ; Kandasamy Arul Kumar ; Latha Mala Priyadhershini Murugaiyan ; Dhinakarraj Gopal ; Raman Muthusamy
- Source: IET Nanobiotechnology, Volume 14, Issue 1, p. 14 –18
- DOI: 10.1049/iet-nbt.2019.0187
- Type: Article
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In this study, chlorhexidine (CHX)–silver (Ag) hybrid nanoparticles (NPs) coated gauze was developed, and their bactericidal effect and in vivo wound healing capacities were tested. A new method was developed to synthesise the NPs, wherein Ag nitrate mixed with sodium (Na) metaphosphate and reduced using Na borohydride. Finally, CHX digluconate was added to form the hybrid NPs. To study the antibacterial efficacy of particles, the minimal inhibition concentration and biofilm degradation capacity against Gram-positive and Gram-negative bacteria was studied using Escherichia coli and Staphylococcus aureus. The results indicated that the NP inhibited biofilm formation and was bactericidal as well. The gauze was doped with NPs, and its wound healing property was evaluated using mice model. Results indicated that the wound healing process was fastened by using the NPs gauze doped with NPs without the administration of antibiotics.
- Author(s): Nahid Ghorbanzadeh ; Amir Peymani ; Hossein Ahmadpour-Yazdi
- Source: IET Nanobiotechnology, Volume 14, Issue 1, p. 19 –24
- DOI: 10.1049/iet-nbt.2019.0088
- Type: Article
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Ureaplasma urealyticum (uu) is one of the most common agents of urogenital infections and is associated with complications such as infertility, spontaneous abortion and other sexually transmitted diseases. Here, a DNA sensor based on oligonucleotide target-specific gold nanoparticles (AuNPs) was developed, in which the dispersed and aggregated states of oligonucleotide-functionalised AuNPs were optimised for the colorimetric detection of a polymerase chain reaction (PCR) amplicon of U. urealyticum DNA. A non-cross-linking approach utilising a single Au-nanoprobe specific of the urease gene was utilised and the effect of a PCR product concentration gradient evaluated. Results from both visual and spectral analyses showed that target–Au-nanoprobe hybrids were stable against aggregation after adding the inducer. Furthermore, when a non-target PCR product was used, the peak position shifted and salt-induced aggregation occurred. The assay's limit of detection of the assay was 10 ng with a dynamic range of 10–60 ng. This procedure provides a rapid, facile and low-cost detection format, compared to methods currently used for the identification of U. urealyticum.
- Author(s): Babli Debnath ; Sumit Sarkar ; Ratan Das
- Source: IET Nanobiotechnology, Volume 14, Issue 1, p. 25 –32
- DOI: 10.1049/iet-nbt.2019.0161
- Type: Article
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In this study, saponin capped triangular silver nanocrystals have been synthesised using fenugreek seed extract, where the extract acts both as a reducing and capping agent. X-ray diffraction study confirms the purity and crystalline nature of the prepared nanocrystals and transmission electron microscopic study shows the triangular morphology with the average edge length of 72 nm, along with the atomic force microscopy study for the height or the width of the triangular nanocrystals. These nanocrystals have been investigated against a few pulses (seeds) such as Pisum sativum, Cicer arietinum and Vigna radiata for their effect on the germination as well as growth of root and shoot. Considering different concentration of silver nanocrystals solution, it has been found that 25 × 10−4 and 80 × 10−4 μg/ml are the minimum and maximum concentrations of silver nanocrystals, within this range, germination and subsequent growth of root and shoot are effective. The result shows significant positive influence on the growth of root and shoot of all seeds in comparison to those of unexposed control germination. Therefore, the result of this experiment has confirmed that the use of saponin capped silver nanocrystals enhances the germination and growth of plants.
- Author(s): Nada Saleh ; Naveed Ahmed ; Muhammad Imran Asad ; Iqra Chaudhery ; Nasir Mahmoud Ahmad
- Source: IET Nanobiotechnology, Volume 14, Issue 1, p. 33 –39
- DOI: 10.1049/iet-nbt.2019.0148
- Type: Article
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Magnetic manotheranostics can be a fascinating charm to diagnose a tumour with MRI, and treatment through hyperthermia. This study aims to synthesise and characterise magnetically responsive polymer colloids (MRPCs). Healthy tissue damage done by chemotherapy session could be minimised by MRPCs. For the colloidal formulation of MRPCs, the oil in water emulsion technique was employed with the aid of sonication and stirring. The organic phase of emulsion contained methotrexate (MTX) drug, Eudragit E100 and CoFe2O4 (synthesised by co-precipitation) in ethanol, and the aqueous phase contained tween 80 in deionised water. The emulsion was optimised by studying/adjusting two different parameters, i.e. the concentration of constituents and sonication cycles. Multiple formulations were produced at sonication amplitude of 60% at 20 kHz, followed by centrifugation and lyophilisation. Characterisation of MRPCs was done for morphology, size measurement (23–25 nm), surface charge (∼15.12), coercivity (∼1549.6 G), magnetisation (2.6 emu) and retentivity (1.34 emu). Drug release in simulating physiological environment (pH = 7.4), was observed for up to 48 h, and, to determine the best release kinetic mechanism results were compared with kinetic models. Magnetic hyperthermia studies showed that MRPCs achieved an acceptable temperature of 42°C, for hyperthermia treatments in cancer patients.
- Author(s): Avinash P. Ingle ; Rafael R. Philippini ; Mahendra Rai ; Silvio Silvério da Silva
- Source: IET Nanobiotechnology, Volume 14, Issue 1, p. 40 –46
- DOI: 10.1049/iet-nbt.2019.0181
- Type: Article
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The present study demonstrated the preparation of three different acid-functionalised magnetic nanoparticles (MNPs) and evaluation for their catalytic efficacy in hydrolysis of cellobiose. Initially, iron oxide (Fe3O4)MNPs were synthesised, which further modified by applying silica coating (Fe3O4-MNPs@Si) and functionalised with alkylsulfonic acid (Fe3O4-MNPs@Si@AS), butylcarboxylic acid (Fe3O4-MNPs@Si@BCOOH) and sulphonic acid (Fe3O4-MNPs@Si@SO3H) groups. The Fourier transform infrared analysis confirmed the presence of above-mentioned acid functional groups on MNPs. Similarly, X-ray diffraction pattern and energy dispersive X-ray spectroscopy analysis confirmed the crystalline nature and elemental composition of MNPs, respectively. TEM micrographs showed the synthesis of spherical and polydispersed nanoparticles having diameter size in the range of 20–80 nm. Cellobiose hydrolysis was used as a model reaction to evaluate the catalytic efficacy of acid-functionalised nanoparticles. A maximum 74.8% cellobiose conversion was reported in case of Fe3O4-MNPs@Si@SO3H in first cycle of hydrolysis. Moreover, thus used acid-functionalised MNPs were magnetically separated and reused. In second cycle of hydrolysis, Fe3O4-MNPs@Si@SO3H showed 49.8% cellobiose conversion followed by Fe3O4-MNPs@Si@AS (45%) and Fe3O4-MNPs@Si@BCOOH (18.3%). However, similar pattern was reported in case of third cycle of hydrolysis. The proposed approach is considered as rapid and convenient. Moreover, reuse of acid-functionalised MNPs makes the process economically viable.
- Author(s): T. Manikandan ; G. Padmalaya ; S. Mahalakshmi ; A.S. Nivethitha ; V. Pavithran ; S. Perumal ; B.S. Sreeja ; P. Senthil Kumar
- Source: IET Nanobiotechnology, Volume 14, Issue 1, p. 47 –52
- DOI: 10.1049/iet-nbt.2019.0193
- Type: Article
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In this research, the hydrothermal method was used for synthesising cellulose acetate (CA) templated nanorods like CuS fibres using vegetable extract (Brassica oleracea var. italica). These extracts act as a reducing agent in the presence of CA. Surprisingly, when the same reaction was carried out in the absence of CA and broccoli extract, structural morphology disappeared and was found as agglomerated. In the presence of cellulose templated extract mediated CuS has revealed as nanorods like fibres and was confirmed by field emission scanning electron microscope analysis. Their crystallinity property of CuS nanoparticles (NPs) and cellulose templated biosynthesised CuS NPs was analysed and compared using X-ray diffraction technique. The biological activities of the obtained product were examined for antibacterial assessment against two bacterial strains that include two-gram negative strains (E.Coli and S. aureus). The nanostructured product found to exhibit excellent antibacterial agent against all the strains. Biosynthesised nanostructure showed its efficacy against A549 lung cancer cells which might attribute to a larger surface to volume ratio of nanorods like fibres. The authors observation suggest that CuS nanorods like fibres can significantly reduce the cell growth with IC50 value of 31.2 μg/ml.
- Author(s): Xin Li and Ce Guo
- Source: IET Nanobiotechnology, Volume 14, Issue 1, p. 53 –58
- DOI: 10.1049/iet-nbt.2019.0261
- Type: Article
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Insects are one of the most agile flyers in nature, and studying the kinematics of their wings can provide important data for the design of insect-like wing-flapping micro aerial vehicles. This study integrates high-speed photogrammetry and three-dimensional (3D) force measurement system to explore the kinematics of Cyrtotrachelus buqueti during the wing-flapping flight. The tracking point at the wing tip of the hind wing was recorded using high-speed videography. The lift-thrust force characteristic of wing-flapping motion was obtained by the 3D force sensor. Quantitative measurements of wing kinematics show that the wing-flapping pattern of the hind wing of C. buqueti was revealed as a double figure-eight trajectory. The kinematic modelling of the wing-flapping pattern was then established by converting the flapping motion into rotational motion about the pivoting wing base in the reference coordinate system. Moreover, the lift force generated by C. buqueti during the wing-flapping flight is sufficient to support its body weight without the need to use thrust force to compensate for the lack of lift force.
- Author(s): Xizheng Fan and Yanyan Zheng
- Source: IET Nanobiotechnology, Volume 14, Issue 1, p. 59 –65
- DOI: 10.1049/iet-nbt.2019.0188
- Type: Article
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The hydrotalcite-supported palladium (Pd) catalyst is prepared with a green and environmentally friendly route, introducing the extract of withered leaves as a dispersant and reducing agent (Pd/LDHs-B). Compared with the as-prepared catalyst (Pd/LDHs-P with the average diameter of 4.3 nm) using a chemical synthesis method with polyvinylpyrrolidone as a dispersant and ascorbic acid (Vc) as a reductant, the results indicate that the size of Pd nanoparticles in Pd/LDHs-B is smaller (ca. 3.6 nm). The Pd-LHDs-B (0.5 mmol‰) exhibits higher activity (98.66%) than Pd/LDHs-P (98.19%) in the Suzuki reaction of 4-bromotoluene and phenylboronic acid at 60°C for 30 min. Also, the reusability of Pd/LDHs-B is confirmed by recycling tests without a significant decrease in activity.
- Author(s): Wenjing Wang ; Fengzhen Zhou ; Xiyao Cheng ; Zhengding Su ; Huiling Guo
- Source: IET Nanobiotechnology, Volume 14, Issue 1, p. 67 –72
- DOI: 10.1049/iet-nbt.2019.0271
- Type: Article
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To effective capture and universal enrichment of His-tagged protein, polyacrylic acid (PAA) brushes were used to encapsulate Fe3O4 nanoparticles, connect NTA, and Ni2+ to prepare magnetic beads. These materials provide many advantages, such as excellent stability, tuneable particle size, and a surface for further functionalisation with biomolecules. His-tagged green fluorescence protein (GFP) was separated efficiently, and the binding capacity of Fe3O4/MPS@PAA/NTA-Ni2+ was 93.4 mg/g. Compared with High-Affinity Ni-NTA Resin and Ni-NTA Magnetic Agarose Beads, Fe3O4/MPS@PAA/NTA-Ni2+ nanocomposites exhibited higher separation efficiency and binding capacity towards His-tagged GFP. Moreover, the selectivity and recyclability of them for the target proteins were maintained well after six cycles. This study would widen the application of PAA in constructing multifunctional nanocomposites for biomedical fields.
- Author(s): Marziyeh Ranjbar-Mohammadi ; Elham Mousavi ; Mohammad Mostakhdem Hashemi ; Mahdi Abbasian ; Jahanbakhsh Asadi ; Ehsan Esmaili ; Mehrafarin Fesharaki ; Pouyan Asadi ; Zahra Arab-Bafrani
- Source: IET Nanobiotechnology, Volume 14, Issue 1, p. 73 –77
- DOI: 10.1049/iet-nbt.2019.0278
- Type: Article
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In this study, we investigated whether the nanofibers produced by natural-synthetic polymers can probably promote the proliferation of co-cultured adipose-derived stem cells/human fibroblast cells (ADSs/HFCs) and synthesis of collagen. Nanofiber was fabricated by blending gelatin and poly (L-lactide co-ɛ-caprolactone) (PLCL) polymer nanofiber (Gel/PLCL). Cell morphology and the interaction between cells and Gel/PLCL nanofiber were evaluated by FESEM and fluorescent microscopy. MTS assay and quantitative real-time polymerase chain reaction were applied to assess the proliferation of co-cultured ADSs/HFCs and the collagen type I and III synthesis, respectively. The concentrations of two cytokines including fibroblast growth factor-basic and transforming growth factor-β1 were also measured in culture medium of co-cultured ADSs/HDCs using enzyme-linked immunosorbent assay assay. Actually, nanofibers exhibited proper structural properties in terms of stability in cell proliferation and toxicity analysis processes. Gel/PLCL nanofiber promoted the growth and the adhesion of HFCs. Our results showed in contact co-culture of ADSs/HFCs on the Gel/PLCL nanofiber increased cellular adhesion and proliferation synergistically compared to non-coated plate. Also, synthesis of collagen and cytokines secretion of co-cultured ADSs/HFCs on Gel/PLCL scaffolds is significantly higher than non-coated plates. To conclude, the results suggest that Gel/PLCL nanofiber can imitate physiological characteristics in vivo and enhance the efficacy of co-cultured ADSs/HFCs in wound healing process.
- Author(s): Sanchaita Lala
- Source: IET Nanobiotechnology, Volume 14, Issue 1, p. 78 –85
- DOI: 10.1049/iet-nbt.2019.0124
- Type: Article
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The study aims to document the effect of starch-stabilised copper-based nanoparticles (CuNPs) on the biosynthesis of pharmaceutically valuable secondary metabolites, especially saponins, of the reputed nootropic herb Bacopa monnieri (L.) Pennell. CuNPs were synthesised chemically by the reduction of cupric sulphate pentahydrate with ascorbic acid using starch as the capping agent. They were characterised by UV–visible spectrophotometry, Fourier-transform infra-red spectroscopy, X-ray diffraction, high-resolution transmission electron microscopy and zeta potential. The nanoparticles consisted of cuprous oxide and metallic copper, were approximately spherical, polydispersed with diameter <20 nm. Hydroponically grown B. monnieri plants were treated in vivo with the CuNPs between the concentrations of 0–100 mg l−1. Spectrophotometric estimation of the total contents of saponins, alkaloids, phenolics, flavonoids and DPPH radical scavenging capacity from the methanolic extracts of the whole plants showed a hormetic increase in the content of secondary metabolites in a concentration-dependent manner from 5 mg l−1 until it declined at toxic metabolic concentration. This was accompanied by an increase in ROS markers hydrogen peroxide and malondialdehyde as well as a hormetic effect on activities of phenylalanine ammonia lyase and antioxidant enzymes catalase, ascorbate peroxidase and superoxide dismutase. CuNPs at sub-toxic concentrations were found to enhance secondary metabolism and antioxidant capacity in Bacopa monnieri through ROS-mediated defence response.
- Author(s): Virna M. Martín Giménez ; Marcos G. Russo ; Griselda E. Narda ; Lucía B. Fuentes ; Luciana Mazzei ; Carlos Gamarra-Luques ; Diego E. Kassuha ; Walter Manucha
- Source: IET Nanobiotechnology, Volume 14, Issue 1, p. 86 –93
- DOI: 10.1049/iet-nbt.2019.0108
- Type: Article
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Drug encapsulation in nanocarriers such as polymeric nanoparticles (Nps) may help to overcome the limitations associated with cannabinoids. In this study, the authors’ work aimed to highlight the use of electrospraying techniques for the development of carrier Nps of anandamide (AEA), an endocannabinoid with attractive pharmacological effects but underestimated due to its unfavourable physicochemical and pharmacokinetic properties added to its undesirable effects at the level of the central nervous system. The authors characterised physicochemically and evaluated in vitro biological activity of anandamide/ɛ-polycaprolactone nanoparticles (Nps-AEA/PCL) obtained by electrospraying in epithelial cells of the human proximal tubule (HK2), to prove the utility of this method and to validate the biological effect of Nps-AEA/PCL. They obtained particles from 100 to 900 nm of diameter with a predominance of 200–400 nm. Their zeta potential was −20 ± 1.86 mV. They demonstrated the stable encapsulation of AEA in Nps-AEA/PCL, as well as its dose-dependent capacity to induce the expression of iNOS and NO levels and to decrease the Na+/K+ ATPase activity in HK2 cells. Obtaining Nps-AEA/PCL by electrospraying would represent a promising methodology for a novel AEA pharmaceutical formulation development with optimal physicochemical properties, physical stability and biological activity on HK2 cells.
- Author(s): Miguel Ángel Aguilar-Méndez ; Teodoro Espinosa-Solares ; Flor de María Guerrero-Toledo ; Daniel Canseco-González ; Azucena Velázquez-Hernández ; Guadalupe Stefanny Aguilar-Moreno ; Elizabeth Navarro-Cerón
- Source: IET Nanobiotechnology, Volume 14, Issue 1, p. 94 –97
- DOI: 10.1049/iet-nbt.2019.0204
- Type: Article
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Nanoparticles of magnetite passivated with gelatin and starch were synthesised using a co-precipitation technique. The nanoparticles were characterised using ultraviolet–visible (UV–vis), dynamic light scattering (DLS), Zeta potential, transmission electron microscope (TEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). The UV–vis spectra showed characteristic surface plasmon resonance of magnetite nanoparticles. The DLS results showed the nanoparticles to have average hydrodynamic diameters of 138 ± 2 and 283 ± 21 nm for particles passivated with gelatin and starch, respectively. The stability in a colloidal solution was greater in nanoparticles passivated with gelatin than nanoparticles obtained with starch, as can be seen by their Zeta potential value (−31 ± 2 and −16 ± 0.5 mV, respectively). According to the TEM evaluation, the use of gelatin allowed to obtain nanoparticles with a spherical morphology and an average size of 10 ± 2 nm. However, when using starch the nanoparticles exhibited diverse morphologies with an average size of 25 ± 7 nm. The XRD results confirmed the crystalline structure of the samples, which showed crystallite sizes of 14.90 and 24.43 nm for nanoparticles passivated with gelatin and starch, respectively. FTIR analysis proved the establishment of interactions between functional groups of biopolymers and magnetite nanoparticles.
- Author(s): Yunchao Xu ; Yunyi Wang ; Huijin Lin ; Xiaokun Liu ; Zuci Zheng ; Tingyin Wang ; Shangyuan Feng
- Source: IET Nanobiotechnology, Volume 14, Issue 1, p. 98 –104
- DOI: 10.1049/iet-nbt.2019.0274
- Type: Article
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A highly sensitive, non-invasive, and rapid HBV (Hepatitis B virus) screening method combining membrane protein purification with silver nanoparticle-based surface-enhanced Raman scattering (SERS) spectroscopy was developed in this study. Reproducible serum protein SERS spectra were obtained from cellulose acetate membrane-purified human serum from 94 HBV patients and 89 normal groups. Tentative assignments of serum protein SERS spectra showed that the HBV patients primarily led to specific biomedical changes of serum protein. Principal components analysis and linear discriminate analysis were introduced to analyse the obtained spectra, with the diagnostic sensitivity of 92.6% and specificity of 77.5% were achieved for differentiating HBV patients from normal groups.
- Author(s): Vahid Ziaaddini ; Maryam Saeidifar ; Mahboubeh Eslami-Moghadam ; Mandana Saberi ; Masoud Mozafari
- Source: IET Nanobiotechnology, Volume 14, Issue 1, p. 105 –111
- DOI: 10.1049/iet-nbt.2019.0086
- Type: Article
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To sustained release of an anticancer drug, oxaliplatin (OX), a non-toxic and biocompatible nanocarrier based on bovine serum albumin (BSA) were synthesised by desolvation method and characterised using Fourier-transform infrared (FTIR) spectroscopy, field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM) and dynamic light scattering. The results showed that the BSA nanoparticles (BSANPs) with a mean magnitude of 187.9 ± 1.2 nm have spherical morphology with a smooth surface and a uniform distribution. Furthermore, OX was loaded onto the BSANPs and the loading was confirmed by FTIR, AFM and FESEM techniques. The percentage of encapsulation efficiency and drug loading were determined by absorption spectroscopy (UV–vis). The drug release studies showed that release of OX from BSANPs exhibited slower release rate. However, the release kinetics followed the first-order kinetic for both of them with the non-Fickian release behaviour. The electrochemical analysis showed stability of OX loaded onto the BSANPs (OX@BSANPs) and confirmed the diffusion mechanism. Furthermore, the results of MTT assay revealed increasing of normal cell viability and cancer cell death in the OX@BSANPs compared to only OX. It was shown that the BSANPs could be safely used as a biocompatible nanocarrier for the sustained release of OX.
Studies on antimicrobial and wound healing applications of gauze coated with CHX–Ag hybrid NPs
Colorimetric-based detection of Ureaplasma urealyticum using gold nanoparticles
Effects of saponin capped triangular silver nanocrystals on the germination of Pisum sativum, Cicer arietinum, Vigna radiata seeds & their subsequent growth study
Co-encapsulating CoFe2O4 and MTX for hyperthermia
Catalytic hydrolysis of cellobiose using different acid-functionalised Fe3O4 magnetic nanoparticles
Facile hydrothermal bio-synthesis of cellulose acetate templated CuS nanorods like fibres: antibacterial, cytotoxicity effects and DNA cleavage properties against A549 lung cancer cells
Wing-kinematics measurement and flight modelling of the bamboo weevil C. buqueti
Biosynthesis of eco-friendly and recyclable Pd/LDHs catalyst using the withered leaves extract for Suzuki coupling reaction
High-efficiency Ni2+-NTA/PAA magnetic beads with specific separation on His-tagged protein
Efficient co-cultivation of human fibroblast cells (HFCs) and adipose-derived stem cells (ADSs) on gelatin/PLCL nanofiber
Enhancement of secondary metabolites in Bacopa monnieri (L.) Pennell plants treated with copper-based nanoparticles in vivo
Synthesis, physicochemical characterisation and biological activity of anandamide/ɛ-polycaprolactone nanoparticles obtained by electrospraying
Synthesis and characterisation of magnetite nanoparticles using gelatin and starch as capping agents
Serum analysis method combining cellulose acetate membrane purification with surface-enhanced Raman spectroscopy for non-invasive HBV screening
Improvement of efficacy and decrement cytotoxicity of oxaliplatin anticancer drug using bovine serum albumin nanoparticles: synthesis, characterisation and release behaviour
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