A Review on Synthesis of Nanostructured Metal Oxide

Copyright © 2015 by American Scientific Publishers All rights reserved. Printed in the United States of America

Reviews in Advanced Sciences and Engineering Vol. 4, pp. 1–13, 2015 (www.aspbs.com/rase)

A Review on Synthesis of Nanostructured Metal Oxide Powder via Soft Chemical Approach Taimur Athar1, ∗ , Aleem Ahmad Khan2 , Sandeep Vishwakarma2 , Alabass Razzaq1 , and Alqaralosy Ahmed1 1

OBC, CSIR-Indian Institute of Chemical Technology, Hyderabad, Andhra Pradesh 500007, India Salar-E-Millat Research Centre and Central Laboratory for Stem Cell Research and Regenerative Medicine, Centre for Liver Research and Diagnostics, Deccan College of Medical Sciences, Hyderabad, Andhra Pradesh 500058, India 2

ABSTRACT Soft chemical approach helps for the synthesis of colloidal dispersion inorganic materials at relatively low temperatures and with simple set up. With the help of better understanding the donor–acceptor properties relationship leads to the formation of nuclei and its growth mechanism to design self-assembled nanostructured framework at the micro to nanoscale. In this review, synthesis and physico–chemical properties of molecular precursors has been investigated for soft chemical approach for designing a cost effective monodisperable crystalline functional material with reproducible yield having high specific surface area–functional properties relationship. A mechanistic approach based on “lock-key molecular interaction” helps to establish new synthetic synergism relationship with high productivity and ultra-purity with their well-defined functional M–O–M framework via self assembly. Molecular self-assembled structure help to understand the tailored composition– structure–properties relationship. The biological safety of such material needs to be assessed by using standard tests at the cellular and molecular level. The results shows most of the metal oxide nanopowder were non-toxic in the biological systems. KEYWORDS: Metal Oxide Frameworks, Nanostructured Composition-Structure-Properties Relationship.

CONTENTS 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Methodologies to MOx Nanopowder . . . . . . . . . . 3. Analytical Techniques Used for Characterization of MOx Nanoparticles . . . . . . . . . . . . . . . . . . . . . 4. Safety Aspects of Nanoparticles . . . . . . . . . . . . . 5. Assessment of Cell Membrane Integrity Assessment Using Fluorescein Diacetate (FDA) . . . . . . . . . . . 6. LDH Activity Measurement to Assess Cytolysis . . . 7. Assessment of Cellular Metabolic Activity by Mitochondrial Oxidation . . . . . . . . . . . . . . . . . . 8. Ureagenesis: A Functional Detoxification Assay . . . 9. Optical Imaging of Nanoparticles Internalized Inside the Cell . . . . . . . . . . . . . . . . . . . . . . . . 10. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . References and Notes . . . . . . . . . . . . . . . . . . . .

....... .......

1 4

....... .......

7 8

....... .......

8 9

....... .......

10 10

....... ....... .......

11 12 12

1. INTRODUCTION The synthesis of colloidal monodispersable nanoparticles with favorable parameters remains a synthetic challenge ∗

Author to whom correspondence should be addressed. Email: [email protected] Received: 3 March 2015 Accepted: xx Xxxx xxxx

Rev. Adv. Sci. Eng. 2015, Vol. 4, No. 3

Growth,

Molecular

Interaction,

Tailored

as shown in Figure 1. Soft chemical approach helps for the synthesis of inorganic materials with colloidal dispersion at relatively low temperatures and with simple apparatus set up. By better understanding the donor–acceptor properties relationship leads to the formation of nuclei and its growth mechanism to design self-assembled nanostructured framework from microlevel to nanoscale.1–4 The structure and composition of nanopowder depend with the preparation condition, such as the nature of the precursors, the role of metal ion source, temperature and finally pH. It offers many advantages such as: (i) tailormade materials can be carried out with good process control, (ii) homogeneous multicomponent systems due to mixing in the solvent, (iii) low temperature for materials processing.5–10 The main parameters that influence the kinetics, growth reactions, hydrolysis, condensation reactions, and consequently, the structural properties have been highlighted. Soft chemical approach helps to enhance our understanding with “origins” of key properties for everyday materials and its structures. The synergic and balanced information based on synthesis–structural properties help to fabricate the nanodevices with high reliability and

2157-9121/2015/4/001/013

doi:10.1166/rase.2015.1098

1

A Review on Synthesis of Nanostructured Metal Oxide Powder via Soft Chemical Approach

Athar et al.

Taimur Athar is working as senior Principal scientist in Indian Institute of Chemical Technology, Hyderabad. Field of specialization: Material science, Sol–gel, Metal Alkoxide, structural characterization and Structural-properties relationship. Research Theme: Molecular chemistry → solid state chemistry → Nanoscience

Aleem Ahmad Khan is a leading scientist in Hepatic Stem Cell Research. He has been a pioneering researcher since many years in the area of hepatocytes transplantation for the treatment of liver cirrhosis. Presently, he is working as Senior scientist and Associate Professor at Salar-E-Millat Research Centre and Central Laboratory for Stem Cell Research and Regenerative Medicine, CLRD, Deccan College of Medical Sciences, Hyderabad. Field of specialization: Stem Cells and Regenerative Medicine, Transplantation Immunology, Nanobiotechnology, Bioartificial organ development, Liver Regeneration, Diabetes cell therapy, Neural regeneration, Cell and molecular biology.

Sandeep Vishwakarma is an eminent Research Scientist at Central Laboratory for Stem Cell Research and Regenerative Medicine, CLRD, and Salar-E-Millat Research Centre, Deccan College of Medical Sciences, Hyderabad. Field of specialization: Stem Cells and Translational Medicine, Nanobiotechnology, Neo-organogenesis, Developmental disorders, Molecular genetics, Cell therapy in neurodegenerative diseases.

Alabass Razzaq is a M.Pharm. He has done project in drug delivery system using nanoparticles.

Alqaralosy Ahmed is M.Tech. student. He has done the project in ceramic materials.

2

Rev. Adv. Sci. Eng., 4, 1–13, 2015

Athar et al.


Synthon

Functional properties Mono disperable nano powder

Surface morphology

Stability

Synthetic Challenge

Structural properties

Synthetic

Molecular Precursor

Soft Chemical Approach

Industrial Scale Up

Application

Rev. Adv. Sci. Eng., 4, 1–13, 2015

Organic transformation Use

Fig. 3.

Catalyst leads

Sensors

Fig. 2. Different approach used for the synthesis of functional metal oxide nanopowder at moderate temperature.

Characterisation

Nanomaterials structure-composition properties

Fig. 1. With required properties—A synthetic challenge for functional ultrapure nanomaterials.

accuracy. By refining our knowledge in surface chemistry help to understand the structural parameters by controlling thermodynamic-kinetic parameters at the bench scale to the production level.11–16 Apart from the quantum effect-surface effect, the bottom up assembly concept has overcome conventional top down material synthetic methods. Nano-structures have the ability to generate new features and perform new functional activities due to their novel physical/chemical properties. At nanoscale, the surface properties become more dominant with their functional properties.11–16 The electronic structure of materials becomes size-dependent which help to understand the structural behavior at the micro/nanoscale to improve functional properties. The review will highlight the state of art for the cost effective synthesis of ultra-pure functional nanomaterials from single source molecular precursor, to understand the structural-functional–surface properties relationship with the help of Lewis acidic behavior of the metal ion involved in the four types of synthetic process as shown in Figure 2 with graphical theme as follows in Figure 3. The chemistry from molecular precursors with their microstructure properties into final nanomaterials with new functional properties has not yet well established in longterm thermal stability and structural morphology. With better understanding the fundamental principles of soft chemical approach help in synthesis of metal oxide materials with desired morphology remains a big synthetic challenge in nanochemistry.11 12 The review will emphasize to

Soft chemical Approach

Properties harvesting

Use

Graphical theme of the review.

understand its applications to elaborate an efficient way to handle agglomerated free nanosized materials at low temperature. The interaction between single source inorganic precursors is the key factor to design nanomaterials with desired functional properties for their future use in nanotechnologies. The change in chemistry from molecular precursor towards the synthesis of nanomaterial with new physical–chemical–mechanical properties depends with change in coordination behavior of central metal ion and quantum confinement of electron in nanometer sized structure which help in better understanding the particle morphology to design technological grade nanomaterials at low cost with high reliability and accurate sensitivity.15–26 Therefore the focus in the review as follows as shown in Figure 4. 1. Based on the principle of molecular structure design concept (MSDC) which depends in principle with number of donor atom and its steric protection around the central metal ion help to design alkoxy based precursor. 2. Synthesis of multifunctional Inorganic materials with scale up for production retaining right stoichiometric ratio and help to better understand the structural-surface– morphology properties relationship. 3. Eco-friendly approach helps to build up hierarchical architecture with spatial arrangements. 4. The chemical transformation from molecular precursor towards metal oxide nanopowder gives uniform size with narrow distribution due to molecular movement and energy distribution due to different crystal planes with growth of different nuclei. However the stable crystal nuclei with good dispersion help to reduce super saturation which helps to inhibit a secondary nucleation and growth of crystal nuclei. However, two general procedures were carried out for the synthesis of nanomaterials, but the physical method has limited properties. The cost effective chemical approach is an efficient method for the design of functional nanoparticles as highlighted in Figure 5 along with advantages as shown in Figure 6. 3


Synthesis of Inorganic materials

Soft chemical Approach

Athar et al.

parameters

Molecular Precursor

Colloidal self assembly with

characterization Structural-physio-chemical functional-property relationship

Applications Broad outline of the review

Precursor chemistry particle chemistry physicochemical properties

Applications

The mode of write-up For nanomaterial

Stereo chemistry Molecular unit

Controlling factor

functionality

Assembly

Reactivity good Stablity

Physical properties

Soft chemical approach Precursor

Nucleation growth Aggregation

Metal Oxide nanopowder

final Material

Mechanical properties

Surface Properties

Chemical composition Phase composition Morphology Porosity

Fig. 4. Syngeric reaction between precursor with characteristic functional properties in metal oxide nanopowder.

2. METHODOLOGIES TO MOx NANOPOWDER Synthesis of molecular framework with an efficient self assembly depends on fundamental understanding of molecular precursor along with their components. The structural-physico–chemical properties relationship help to design the nanobuilding block with refined surface properties. Synthesis and characterization of molecular precursor will be carried out to prepare24–38 (1) ultra-pure nanosized functional materials by controlling properties at moderate temperature by retaining compositional uniformity intact. 4

(2) To prepare monodisperse and well-defined technological grade nanomaterials with fine tuning of chemical and microstructural properties from micro to nanoscale, help to study in depth structural-reactivity properties relationship with better understanding “Design Rule” to enhance functional performance. (3) To improve an environmental compatibility with green synthetic approach, which depends on reaction time, temperature and the polarity of the solvents. (4) To undertake detailed study for integrating a nanodevices with reference to particle size. The synergic interaction between single source inorganic molecular precursors is the key factor to design Rev. Adv. Sci. Eng., 4, 1–13, 2015

Athar et al.


Ball milling Combustion synthesis Dry synthesis Physical method

Flame synthesis Plasma synthesis

Controlled Not Nanotechnologies

Particle Properties Under Controlled Soft reaction

Wet Synthesis Chemical method

Hydro/solvothermal synthesis Fig. 5.

Various techniques used for the synthesis of technology oriented nanomaterials.

nanomaterials with desired functional properties for their future use in nanotechnologies as shown below as shown in Figure 7.28–31

efficiency based on structural-reactivity-functional properties remains a synthetic challenge. Table I a

Solvent

M(OR)x

MM OR3 2 + 3MCl2 → MM2 M3 O6 + 6RCl ↑

M(OH)x + (ROH)x

OR

M MOR3 2 + H2 O → M M2 O6 + 6ROH + 3H2 M = Be Mg Ca Sr Ba

300 ºC 450 ºC MO or MO2 Nanocrystalline Powder

Hard chemical reaction

R = CH3 C2 H5 C3 H7n i C4 H9n t

Nanostructured Powder

Molecular precursor

Soft-chemical approach

Leads

b

Controls

Help to Study

MCl2 + 3KOR → KMOR3 + 2KCl 3KMOR3 + M Cl3 → M MOR3 3 + 3KCl M MOR3 3 + 9H2 O → M M3 O9 · H2 O + 9ROH + 5H2

Coarse grained agglomerated structure limited surface area Inhomogenous microstructure Uncontrolled stoichiometry thermodynamic unstable compounds Surface structure composition oxidation state of metalion

M = CoII NiII CuII Zn

M = Ge Sn Ba Sr Ca

M(OH)x

The comparative properties between mono and bimetallic nanopowder have been illustrated in Figure 8. 1. The synthesis and characterization of metal alkoxides between p, d and f type orbital vice-versa and its conversion into corresponding nanomaterials for improving the

Fig. 6.

MCl2 + 2KM OR3 → 2KCl ↓ +MM OR3 2

Helps

role of metal ion controlling factor with self-assembly Design of nanostructured framework

Improves

Size & Shape Composition Morphology Selectivity

Soft chemical reactions

to design

Mono disperse nanomaterials with purity

depends functional materials with tailored properties

Advantages of hard-soft chemical approach.

Rev. Adv. Sci. Eng., 4, 1–13, 2015

5


Table I. Synthesis of bimetal oxide nanoparticle.39–48

Geometric New precursor dependson

Properties of particles depends on following parameters

Electronic Chemical

Fig. 7. The syngeric condition between molecular precursor with their properties.

M = Be Mg Ca Sr Ba

M = Ce Eu Er Yb Ga Al In R = CH3 C2 H5 C3 H7n i C4 H9n t

MCl2 + 3KOR → KMOR3 + 2KCl

c

4KMOR3 + M Cl4 → M MOR3 4 + 4KCl M MOR3 4 + 12H2O → M M4 O12 + 12ROH + 6H2 M = Be Mg Ca Sr Ba

M = Ti Zn Sn Ge

R = CH3 C2 H5 C3 H7n i C4 H9n t d

MCl3 + 3KM OR4 → 3KCl ↓ +MM OR4 3 MM OR4 3 + 3MCl2 → 6RCl ↑ +MM M OR2 O2 3

Molecular precursor i

Er{Al(OPr 4 }3 Er{Ti(OPri 5 }3 Er{Si(OR)5 }3 Er{Si(OR’)5 }3 Er{As(OR)4 }3 Er{As(OR’)4 }3 Er{Sb(OR)4 }3 Er{Sb(OR’)4 }3 Er{Ti(OR)5 }3 Er{Zr(OR)5 }3 Er{Yb(OR”)4 }3 Er(OR”)4 P(OR”)2 Er(OR”)4 P{Yb(OR”)4 }2 Ce{Sb(OPri 4 }3 Ce{Ti(OPri 5 }3 Ce{Sn(OPri 5 }3 Er{Zr(OPri 5 }3 Er{Al(OPri 4 }3 Er{Ti(OPri 5 }3 Cu{Al(OPri 4 }2 Ni{Sb(OPri 4 }2 GdCl3 :Al(OH)3 Ni(OH)2 :SnCl2 Fe(OH)3 :AlCl3 FeCl3 : GdCl3 ZrOCl2 8H2 O:SnCl2 · 2H2 O LaCl3 :Ba(OH)2 Al(OH)3 :ZnCl2 ZnCl2 :Ba(OH)2

MM OR4 3 + 6MCl2 → 12RCl ↑ +MM M2 O4 3 MM OR4 3 + 12H2O→MM3 O12 + 12ROH + 6H2 M = Ce Eu Er Yb

M = ln Tl Bi Al Ga

M = Co Ni Cu Zn

e

M MOR4 3 + 3MCl4 → M M MO4 3 + 12RCl ↑ M = Ce Eu Tb Er Yb M = As Sb Ga ln Fe Al

MM OR4 2 + 2M Cl2

M = Ge Sn Ti Zr Hf

→ 4RCl ↑ +MM M OR2 O2 2

MM OR4 2 + 4M Cl2 → 8RCl ↑ +MM

M2 O4 2

MM OR4 2 + 8H2O → MM2 O8 + 8ROH + 4H2 M = Ce Er Yb

M = Ge Sn Sr Ba Ca R = CH3 C2 H5 C3 H7n i C4 H9n t

Monometallic metal oxide nanoparticles

with

Ce5 Sb3 O13 Ce6 Ti3 O15 Ce6 Sn3 O15 Er6 Zr3 O15 Er5 Al3 O12 Er6 Ti3 O15 CuAl2 O4 NiSb2 O4 Unpublished AlGdO3 Unpublished NiSnO2 Unpublished Fe3 AlO6 Unpublished GdFeO3 Unpublished SnZrO3 Unpublished La2 Ba3 O6 Unpublished Al2 Zn3 O6 Unpublished ZnBaO2 Unpublished

3KMOR4 + M Cl3 → M MOR4 3 + 3KCl ↓

M = Co Ni Cu Zn

Er doped SiO2 –Al2 O3 Er3+ doped SiO2 –TiO2 Er6 Si3 O15 Er6 Si3 O15 Er5 As3 O12 Er5 As3 O12 Er5 Sb3 O12 Er5 Sb3 O12 Er6 Ti3 O15 Er6 Zr3 O15 Er5 Yb3 O12

MCl3 + 4KOR → KMOR4 + 3KCl ↓

f

MCl2 + 2KM OR4 → MM OR4 2 + 2KCl ↓

Nano metal oxides 3+

M MOR4 3 + H2 O → M M3 O12 + 12ROH + 12H2 ↑

R = CH3 C2 H5 C3 H7n i C4 H9n t

Athar et al.

Where: R = CH2 CF3 , R = CH2 CCl3 , R = CH (CF3 2 Nanopowder has great scientific interest due to their uniform properties and it is a link between bulk materials and atomic or molecular structures. The properties were conducted with the help of spectroscopic studies which help to identify both in terms of their electronic and geometric structure. We have emphasized the soft chemical approach synthesis of metal oxide nanopowder with

Melting point differs Electronic structure in Magnetic-catalytic properties Selectivity

Bimetallic with oxide nano particles

Hardness Chemical and Mechanical resistance Thermal stability

Fig. 8. The comparative properties between mono and bimetallic oxide nanopowder.

6

Rev. Adv. Sci. Eng., 4, 1–13, 2015

Athar et al.


controlled particle size. The synthesis of technological grade nanomaterials was carried out by cost-effective wet chemical approach. However, the physical method is not reproducible for the synthesis of technology grade nanomaterials having many surface defective functional properties. The various type of metal oxide nanomaterials as illustrated in Tables I–II. (1) Synthesis and characterization of divalent based metal oxide.

Growth

Stabilization

Nucleation

Structure

Surface Energy

Monodispersity

MCl2 + 2KOR → MOR2 + 2KCl ↓ MOR2 + MCl2 → M2 O2 + 2RCl ↑ Functional Properties

M = Mg Sr Co Ni Cu Zn (2) Synthesis and characterization of trivalent based metal oxide. MCl3 + 3KOR → MOR3 + 3KCl ↓ MOR3 + MCl3 → M2 O3 + 3RCl ↑ M = Eu Tb Dy Yb As Sb Bi Ga Fe (3) Synthesis and characterization of tetravalent based metal oxide. MCl4 + 4KOR → MOR4 + 4KCl ↓ MOR4 + MCl4 → M2 O4 + 4RCl ↑ M = Ti Zr Hf Si Ge Sn The change in chemistry from molecular precursor towards the synthesis of colloidal nanomaterial with new physical–chemical–mechanical properties depends with change in coordination behavior of central metal ion, to enhance the particle properties with respect to its stability, time and temperature along the properties of metal ion as shown in Figure 9. The functionality of nanomaterials depends on quantum confinement of electron along with Table II. Synthesis of metal oxide nanoparticle.49–73 Metal oxide

Reference number

CeO2 ZrO2 TiO2 CuO Nb2 O5 MnO2 CdO CoO MgO SnO2 NiO Sb2 O3 SrO ZnO

[50] [49, 52, 56] [51, 52] [53] [55, 62] [57] [57, 65] [58] [60, 68, 73] [61] [64, 69] [66, 71] [67] [59, 70]

Rev. Adv. Sci. Eng., 4, 1–13, 2015

Fig. 9. The schematic diagram showing the interrelationship for nanomaterials.

surface energy and geometrical approach which helps to control the electrical, optical, magnetic and thermoelectric properties at low temperature with less processing time and minimum cost as shown in Figure 10.28–45 Synthesis of monometallic oxide nanopowder and bimetallic oxide along with graphical profile regarding the progress of reaction has been illustrated in Figures 11 and 12.

3. ANALYTICAL TECHNIQUES USED FOR CHARACTERIZATION OF MOx NANOPARTICLES The characterization depends with the state of the art instrumental and the mode for synthesis of nanostructure which can be used for fabrication of devices. Size, shape and physico-chemical properties of mondisperable nanoparticles are related to their chemical transformations based on synthetic methodologies and its parameters. Many analytical approaches were undertaken for their characterization to study structural properties at the nanoscale as shown in Table III. • Ionization takes place by photons and electrons followed by mass spectra for their analysis by means of quadrupole and time-of-flight mass spectrometers; • Atomization and selection of neutral clusters takes place with respect to their masses. • Transmission and scanning electron microscopes helps to predicts information regarding Size/shape of the Increase Minimize

Nanodevice

Sensitivity Cost

Simple

Scale-up process

Extension

Lower and Upper detection Limit

New Methodology via soft chemical approach

Fig. 10. The co-relationship between synthetic approaches with its application in nanodevices.

7


Mono dispersed colloidal particle Nucleation

Table III. Techniques used to study structural-property relationship and for its characterizations. Properties to be analyzed74–90

Method FT-IR Nucleation Threshold

Injection

Concentration of precursor

Growth

Athar et al.

UV-VIS Spectra

TG-DTA Saturation

Raman Spectra

Time Mass spectroscopy

Fig. 11. Synthesis of monometallic oxide.

particles along with their distribution and topology at the nanoscale. • Electron diffraction helps to study size and phase’s structure with their bond lengths. • STM helps to determine the morphological properties along with their surface area. • Adsorption of gases taken by the particle to gives information about their surface area. • Photoelectron spectroscopy helps to determine the electronic structure of particle. • Conductivity measurement gives information about the conduction band, percolation and topology in nanomaterials.

XRD BET SEM/TEM Light scattering method

Vibrational stretching frequency of Metal-oxygen bond UV absorption of the amorphous gels and crystalline samples were heated at different temperatures Weight loss and thermal effects occurs during their conversion From precursors into their final metal oxides nanopwder Photons scattering and interaction with other molecule to induce Transitions with different energy levels Separating the ionized species with respects to their mass To study an extent of crystallization in the sample Accessible surface area in the particle To study particle shape, size and morphology Particle size distribution

nanoparticles as illustrated in Table IV.

4. SAFETY ASPECTS OF NANOPARTICLES The nanoparticles synthesized by soft-chemical approach were evaluated for their biological safety to utilize in various applications. The safety of nanoparticles was assessed by using standard tests at cellular and molecular levels. Human hepatic cells were used to investigate in vitro cytotoxicity with different type of mono and bi-metal oxide

Cmax

Solute concentration

Supercrititcal saturation

In order to assess the cell membrane integrity, Fluorescein diacetate (FDA) hydrolysis was used to measure enzymatic activity of trans-membrane lipases. Living cells actively converts the non-fluorescent FDA into green fluorescent compound known as “fluorescin,” a sign of membrane interaction with functional cell as shown in Figure 13.91 92 Briefly, human hepatic cells were seeded in a 24 well culture plate with incubation of nanoparticles at 1 mg/ml

Nucleation and Growth leads to monodispersity Nucleation ceases

Cmin

Table IV. Mono and bimetal oxide nanoparticles synthesized and tested for their in vitro cytotoxicity in human hepatocytes. Cs

Growth leads to polydispersity Particle growth stop

saturated solubility I

II

III

Time Fig. 12. Once nucleation starts the progress of growth takes place as illustrate.

8

5. ASSESSMENT OF CELL MEMBRANE INTEGRITY ASSESSMENT USING FLUORESCEIN DIACETATE (FDA)

Mono metal oxide nanoparticles ZnO (59, 70) SnO2 (Unpublished) Sb2 O3 (66, 71) SrO (67) NiO (64, 69)

Bi metal oxide nanoparticles Fe3 AlO6 (Communicated) FeAl3 O6 (Unpublished) SnZnO3 (Unpublished) Al2 Zn3 O6 (Unpublished) La2 Ba3 O6 (Unpublished) ZnBaO2 (Unpublished) GdAlO3 (Unpublished) GdFeO3 (Unpublished)

Rev. Adv. Sci. Eng., 4, 1–13, 2015

Athar et al.


Fig. 15. activity.

Fig. 13.

Chemical basis of the FDA cytotoxicity assay.

concentration. The effect for cellular viability was evaluated after 72 h by using fluorescence imaging by Carl Zeiss microscope at 40× magnification. The cell viability was established by the ratio between viable (green) and dead (no fluorescence) cells counted on several microscopic fields. Except ZnO and ZnBaO2 none of the mono or bi metal oxide nanoparticles showed any significant cytotoxicity in human hepatocytes as shown in Figure 14.

6. LDH ACTIVITY MEASUREMENT TO ASSESS CYTOLYSIS Lactate dehydrogenase (LDH) assay is a reliable colorimetric assay to quantitatively measure LDH released into the media from damaged cells which can be used as a biomarker for cellular cytotoxicity and cytolysis. LDH is a cytosolic enzyme that is an indicator of cellular toxicity. The assay is ideal parameter for high-input screening and

Fig. 14. FDA (cell membrane integrity assay) showing green fluorescence representing utmost membrane integrity in both the (A) control and (B) nanoparticles treated cells (scale bar: 10 m). Percentage of viable cells stained with FDA was similar to control. ZnO and ZnBaO2 were found to be non-toxic towards cell 0 05) except ZnO and ZnBaO2 which showed statistically significant decreased metabolic activity at ≥50 g/ml concentrations (∗ p < 0 01, ∗∗ p < 0 001, ∗∗∗ p < 0 0001).

reader (BIORAD). Graph plotted between mean values of absorbance for all the variables and nanoparticles concentrations did not show any significant difference in LDH release showing lack of cytotoxicity as well as cytolysis as shown in Figure 16.

Ureagenesis is an important detoxification functional assay to assess the detoxification of Ammonia in liver cells. Removal of ammonia is routinely done by functional hepatocytes in the liver and is performed by five urea cycle enzymes. Failure to this mechanism leads to accumulation of Ammonia causes derrangement basic functions of hepatocytes. In clinical condition hyperammonia leads to changes in blood to brain transport of amino acids and glutamate and the brain energy metabolism which leads to hepatic coma (hepatic encephalopathy). Thus removal of ammonia is an important facet of functional hepatocytes.97 Present study deals with the safety aspects

Fig. 18. Urea cycle in human hepatocytes.

10

Rev. Adv. Sci. Eng., 4, 1–13, 2015

Athar et al.


of the nanoparticles important detoxifying functional assay was performed by using NH4 Cl as a source of NH3 in nanoparticles exposed human hepatocytes as further supported in Figure 18. Briefly, six million human hepatocytes were incubated with different nanoparticles with variable concentrations for 72 h and used to estimate the efficiency of Ammonium chloride detoxification. 2.5 mM NH4 Cl was used to assess the rate of ureagenesis both to control (hepatocytes) and then treated cells (hepatocytes with nanoparticles). No change in urea production was observed in tested nanoparticles except ZnO and ZnBaO2 which showed decreased the urea formation at ≥50 g/ml concentration as shown in Figures 19–21.

9. OPTICAL IMAGING OF NANOPARTICLES INTERNALIZED INSIDE THE CELL

Fig. 20. (A) Identification of nanoparticles by using a conventional microscope. Single nanoparticle produces an optical ring around it and increases its resolution power of 1000 times larger than its nominal size (white arrow). Self-assembly of nanoparticles increases the resolution and appears in rod-shape with increases resolution (yellow arrow, Scale bar: 10 m).

To identify the optical signatures of nanoparticles human hepatic cells were incubated with nanoparticles at 4 C for 2 h internalized in human hepatic cells. Nanoparticles were visualized by using an inverted conventional microscope (Carl Zeiss, Germany) in transmission mode. A 40×

objective lens with a numeric aperture of 0.4 m was used with a high resolution (1324 × 1024) 16-bit monochrome cooled CCD camera. Nanoparticles generated characteristic optical rings (approximately 5 m–10 m) under

Fig. 19. In vitro detoxification functional assay showed that the amount of urea production was similar to the control (untreated) in human hepatic cells with different concentrations of nanoparticles after 72 h of incubation. No statistically significant difference was observed at different concentrations of nanoparticles tested (n = 3, p > 0 05) except ZnO and ZnBaO2 which showed statistically significant decreased ureagenesis at ≥50 g/ml concentrations (∗ p < 0 01, ∗∗ p < 0 001, ∗∗∗ p < 0 0001).

Fig. 21. (B) Internalization of nanoparticles in human hepatocytes (after 2 h of incubation) was clearly observed under 40× objective of a conventional inverted microscope in closed 0.4 m aperture (Scale bar: 10 m). White arrows indicate internalized nanoparticles within the cell. Cells without nanoparticles (control) did not show any signature in conventional microscope and were found difficult to track due to low resolution in closed aperture (Scale bar: 10 m).

Rev. Adv. Sci. Eng., 4, 1–13, 2015

11


inverted microscope and enhanced its resolution more than 1000 times as shown in Figure 22.

10. CONCLUSION Soft chemical approach gives efficient, cheap, robust and active metal oxide nanopowder by controlling the kinetic energy which help to link synthesis–structure properties relationship for determining the morphology–functional properties in nanopowder. With the help of surface morphology, geometry at the nanoscale lead to reactions with better understanding to design a controlled functional nanodevice with activity, structural, selectivity and reliability relationship based on size, shape, chemical composition, geometry and oxidation state of the metal ion. Metal oxide prepared with the help of soft templates which is suitable for large scale up production. All nanoparticles were tested for their cytotoxicity in human hepatocytes were found to have high degree of biocompatibility till 1 mg/ml concentrations except ZnO and ZnBaO2 (≥50 g/ml) showed toxicity ≥50 mg/ml concentration. Identification of nanoparticles using conventional microscopy provides a new dimension for easy labeling and tracking of the cells. Both the structural and functional parameters successfully demonstrated for its use as non-toxic nanoparticles for labeling and tracking of transplanted cells in animal models for cell fate determination.

References and Notes 1. J. A. Rodriguez and M. F. Garcia, Synthesis, Properties and Application of Oxide Nanomaterials, John Wiley and Sons, Hoboken, New Jersey (2007), (ISBN: 978-0-471-72405-6). 2. J. P. Jolivet, Metal Oxide Chemistry and Synthesis, From Solution to Solid State, Wiley Chichester (2000), (ISBN: 978-0-471-97056-9). 3. C. C. Koch, Nanostructured Materials, William Andrew Publishing, New York (2002), (ISBN-10: 0-8155-1354-0-08154). 4. P. Yang, The Chemistry of Nanostructured Materials, World Scientific (2003), ISBN: (130-978-981-4313-06-3) 5. D. L. Fedlheim and C. A. Foss, Metal Nanoparticles, CRC Press (2001), (ISBN: 9780824706043). 6. G. Cao, Nanostructures and Nanomaterials, Imperial College Press (2004), (ISBN-13: 978-1860944802). 7. (a) D. Vollath Nanomaterials, Wiley (2008), (ISBN: 978-3-52733379-0); (b) J. N. Lalena and D. A. Cleary, Principles of Inorganic Materials Design, Wiley New York (2010), (ISBN: 978-0-47040403-4). 8. A. K. Bandyopadhyay, Nanomaterials, New Age International (2007), (ISBN: 978-81-224-2009-8). 9. L. M. Marzan and P. V. Kamat, Nanoscale Materials, Springer (2003), (ISBN: 978-0-306-48108-6). 10. P. Sapra and D D. Sarma, The Chemistry of Nanomaterials: Synthesis Properties and Application, edited by C. N. R. Rao, A. Muller, and A. K. Cheetham, Wiley, VCH, Weenheem (2004), (DOI: 10.1002/352760247X.ch11). 11. S. Mathur, H. Shen, and H. S. Nalwa, Encyclopedia of Nanoscience and Nanotechnology 4, 131 (2004), (ISBN: 1-58883-159-0). 12. K. J. Klabunde and R. M. Richards, Nanoscale Materials in Chemistry, 2nd edn., Wiley, New York (2012), (ISBN: 978-0-47022270-6).

12

Athar et al.

13. A. Muller, A. K. Cheetham, and C. N. R. Rao, Nanomaterials Chemistry: Recent Developments and New Directions, Wiley-VCH (2007), (DOI: 10.1002/352760247X.). 14. J. L. G. Fierro, Metal Oxide: Chemistry and Applications, Taylor and Francis (2005), (ISBN: 9780824723712). 15. U. Schubert and N. Hüsing, Synthesis of Inorganic Materials, WileyVCH-Weinheim (2000), (ISBN: 3-527-29550-X). 16. T. Athar, Metal oxide nanopowder, Emerging Nanotechnologies for Manufacturing, William Andrews InC., edited by W. Ahmad and M. Jackson, 13, Eaton Avenue, Norwich, NY (2008), 13818 (ISBN: 978-0-8155-1583-8). 17. D. C. Bradley, R. C. Mehrotra, and D. P. Gaur, Metal Alkoxides, Academic Press, London (1978), (ISBN: 0121242501). 18. N. Y. Turova, E. P. Turevslaya, V. G. Kessler, and M. I. Yanovskaya, The Chemistry of Metal, Alkoxides, Kluwer, London (2002), (ISBN 978-0-306-47657-0). 19. D. C. Bradley, R. C. Mehrotra, I. P. Rothwell, and A. Singh, Alkoxo and Aryloxo Derivatives of Metals, Academic Press, London (2001), (ISBN-10: 0-12-124140-8). 20. R. C. Mehrotra, R. Bohra, and D. P. Gaur, Metal -diketonates and Allied Derivatives, Academic Press, London (1978), ISBN: (01248815059780124881501). 21. R. C. Mehrotra and A. Singh, Chem. Soc. Rev. 25, 1 (1996). 22. J. D. Mackenzie and E. P. Bescher, Acc. Chem. Res. 40, 810 (2007). 23. D. C. Bradley, Chem. Rev. 89, 1317 (1989). 24. J. Livage, M. Henry, and C. Sanchez, Prog. Solid State Chem. 18, 259 (1988). 25. V. G. Kessler, S. Gohil, and S. Parola, Dalton Trans. 4, 544 (2003). 26. V. G. Kessler, Chem. Commun. 11, 1213 (2003). 27. S. Szafert, J. L. Utko, and P. Sobota, Dalton Trans. 46, 6509 (2008). 28. K J. Edler, Sol–Gel Processing, Kluwer, London (2004), (ISBN: 1-4020-7969-9). 29. L. C. Klein, Sol–Gel Technology for Thin Film, Fibres, Performs, Electronic, and Specialit Shape, Noyes Publications, Mill Road, Park Ridge, New Jersey, USA (1988), (ISBN: 0-8155-1154-X). 30. S. Sakka (ed.), Hand Book of Sol-gel Science and Technology: Processing, Characterization and Applications, Kluwer Academic Publishers Norwell, USA (2004), (ISBN: 1-4020-7966-4). 31. C. J. Brinker and G. W. Scherer, Sol–Gel Science: The Physics and Chemistry of Sol–Gel Processing, Academic Press, San Diego (1990), 108 (ISBN: 0-12-134970-5). 32. T. Athar, R. Bohra, and R. C. Mehrotra, Main Group Metal Chemistry 10, 399 (1987), (ISSN: 0334-7575). 33. T. Athar, R. Bohra, and R. C. Mehrotra, Synth. React. Inorg. Met. Org. Chem. 19, 195 (1989). 34. T. Athar, R. Bohra, and R. C. Mehrotra, Ind. J. Chem. 28, 302 (1989). 35. T. Athar, R. Bohra, and R. C. Mehrotra, Ind. J. Chem. 28, 492 (1989), (ISSN: 0376-4699). 36. T. Athar, R. Bohra, and R. C. Mehrotra, J. Ind. Chem. Soc. 66, 189 (1989), (ISSN: 0019-4522). 37. T. Athar, R. Bohra, and R. C. Mehrotra, J. Ind. Chem. Soc. 67, 535 (1990), (ISSN: 0019-4522). 38. T. Athar, J. O. Kwon, and S. I. Seok, Applied Organometallic Chem. 19, 964 (2005), (ISSN:0268-2605). 39. T. Athar, J. O. Kwon, and S. I. Seok, J. Solid State Chem. 178, 1464 (2005), (ISSN: 0022-4596). 40. T. Athar, J. O. Kwon, and S. I. Seok, Inorganic Chimica Acta 358, 4541 (2005). 41. M. A. Lim, T. Athar, S. I. Seok, W. J. Chung, J. J. Ju, M. H. Lee, and S. I. Hong, J. Mater. Sci. 41, 1285 (2006), (ISSN: 0022-2461). 42. T. Athar, J. O. Kwon, and S. I. Seok, Chin. J. Chem. 25, 998 (2007), (ISSN: 1001-604X). 43. M. A. Lim, T. Athar, S. I. Seok, W. J. Chung, J. J. Ju, M. H. Lee, and S. I. Hong, J. Amer. Ceram. Soc. 90, 116 (2007), (ISSN: 0002-7820). Rev. Adv. Sci. Eng., 4, 1–13, 2015

Athar et al.


44. Z. A. Ansari, A. A. Khan, H. Fouad, T. Athar, and S. G. Ansari, Sensor Letters 12, 1203 (2014). 45. T. Athar, Z. A. Ansari, A. A. Khan, H. Fouad, and S. G. Ansari, Arabian Journal of Chemistry (submitted). 46. T. Athar, Advanced Material Letter 6, 265 (2015), IJNBM: 2009. 027711. 47. T. Athar, Int. J. Nano and Biomaterials 2, 173 (2009), (ISSN: 17528933). 48. T. Athar, S. Han, T. Ko, and I. M. Lee, Synthesis and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chemistry 39, 266 (2009), (ISSN: 1553-3182/3174). 49. T. Athar and K. R. Reddy, Chin. J. Chem. 26, 751 (2008), (ISSN: 1001-604X). 50. T. Athar, K. S. Han, S. Han, T. Ko, and I. M. Lee, Synthesis and Reactivity in Inorganic, Metal-Organic, and Nano-Metal Chem. 39, 261 (2009), (ISSN:15533182/3174). 51. T. Athar, K. S. Han, S. Han, T. Ko, I. M. Lee, Int. Jl. Green Nanotechn.: Materials Science and Engineering 1, M54 (2009), (ISSN:1943-0841). 52. N. Topnani, S. Kushwaha, and T. Athar, Internat. Jl. Green Nanotechn.: Materials Science and Engineering 1, M67 (2009), (ISSN: 1943-0841) 53. T. Athar, Arabian J. Chem. 3, 1319 (2010), (ISSN-1878-5352) 54. N. Suzuki, T. Athar, Y. T. Huang, K. Shiasaki, N. Miyamoto, and Y. Yamuchi, Jl. Ceram. Soc. Jp. 119, 405 (2011). 55. T. Athar, T. Ko, I. M. Lee, and W. Ahmed, Adv. Sci. Lett. 7, 35 (2012), (ISSN 1936-6612). 56. T. Athar, N. Topnani, A. Hakeem, and W. Ahmed, Adv. Sci. Lett. 7, 39 (2012), (ISSN1936-6612). 57. T. Athar, N. Topnani, A. Hakeem, and A. Hashmi, Wet Synthesis of Monodisperse Cobalt Oxide Nanoparticles, Internat. Scholarly Research Network (ISRN)-Material Science (2012), pp. 1–5, (DOI:10.5402/2012/691032). 58. T. Athar, J. Nanoparticles (submitted). 59. T. Athar, N. Topnani, A. Hakeem, and W. Ahmed, Adv. Sci. Lett. 7, 27 (2012), (ISSN 1936-6612). 60. T. Athar, A. Hakeem, and S. G. Ansari, Adv. Sci. Lett. 4, 161 (2012), (ISSN 1936-6612). 61. T. Athar, A. Hashmi, A. Al-Hajry, Z. A. Ansari, and S. G. Ansari, Jl. Nanosci. Nanotechn. 12, 7922 (2012), (ISSN: 1533-4880). 62. T. Athar, A. Hakeem, Z. A. Ansari, and S. G. Ansari, Rev. Adv. Sci. Eng. 2, 77 (2012), (ISSN:2157-9121). 63. T. Athar and A. Hashmi, Mater Focus 2, 263 (2013), (ISSN: 2169429X). 64. T. Athar and A. Hashmi, Mater. Focus 2, 298 (2013), (ISSN: 2169429X). 65. T. Athar and A. Hashmi, Mater. Focus 2, 288 (2013), (ISSN: 2169429X). 66. T. Athar, Mater. Focus 2, 450 (2013), (ISSN: 2169-429X), 67. T. Athar, Mater. Focus 2, 493 (2013), (ISSN: 2169-429X). 68. T. Athar, S. S. M. Shafi, and A. A. Khan, Mater. Focus 3, 378 (2014), (ISSN: 2169-429X). 69. T. Athar, S. S. M. Shafi, and A. A. Khan, Mater. Focus 3, 341 (2014), (ISSN: 2169-429X). 70. T. Athar and S. S. M. Shafi, Adv. Sci. Lett. 20, 1654 (2014), (ISSN 1936-6612). 71. T. Athar, S. S. M. Shafi, and A. A. Khan, Mater. Focus 3, 397 (2014), (ISSN: 2169-429X).

Rev. Adv. Sci. Eng., 4, 1–13, 2015

72. B. M. Choudary, K. Mahendar, L. M. Kantam, K. V. S. Ranganath, and T. Athar, Adv. Synth. Catal. 348, 1977 (2006), (ISSN: 16154150). 73. D. L. G. L. Pavia, G. S. Kriz, and J. R. Vyvyan, Introduction to Spectroscopy, 4th edn., Brooks/Cole Cengage Learning, United State (2009), (ISBN-13: 978-0-495-11478-9). 74. D. A. Skoog, F. J. Holler, and S. R. Crouch, Principles of Instrumental Analysis, 6th edn., Belmont, CA, Thomson Brooks/Cole (2007), p. 169, (ISBN: 0495012017). 75. H. B. Stuart, Infrared Spectroscopy Fundamentals and Applications, Chichester, Eng., J. Wiley, Hoboken, N.J (2004), (ISBN: 978-0-47085428-0). 76. P. Atkins and J. Paula, Physical Chemistry, 8th edn., Oxford University Press, New York (2006). 77. K. Nakamoto, Infrared and Raman spectra of Inorganic and Coordination Compounds, Wiley-Interscience, 6th edn. (2008), (ISBN: 978-0-471-74493-1). 78. A. A. Davydov, Infrared Spectroscopy of Absorbed Species on the Surface of Transition Metal Oxides, Wiley (1990), (ISBN: 047191813X). 79. G. Soacrates, Infrared and Raman Characteristic Group Frequencies: Tables and Charts, John Wiley and sons Ltd. (2001), (ISBN: 10 0470-09307-2). 80. S. Mitra, Sample Preparation Techniques in Analytical Chemistry, John Wiley and sons Ltd., New Jersey (2003), (ISBN: 0-471-32845-6). 81. J. R. Lakowicz, Principle of Fluoresence Spectroscopy, 3rd edn., Springer Science+ Business Media, L L C Baltimore (2006), (ISBN: 978-0-387-46312-4). 82. B. D. Cullity, Elements of X-ray Diffraction, Addison-Wesley Publishing Company, Inc., London (1978), (ISBN: 1178511421, 9781178511420). 83. A. Guinier, X-Ray Diffraction in Crystals, Imperfect Crystals and Amorphous Bodies, Dover publications, New York (1994), (ISBN: 0486680118, 9780486680118). 84. M. P. Klug and L. E. Alexander, X-ray Diffraction procedure for Polycrystalline and Amorphous Materials, Wiley, New York (1974), (ISBN:978-0-471-49369-3). 85. Powder Diffraction Files JCPDS, Ed: International Center for Diffraction Data, Pasadena, CA (1993). 86. I. M. Watt, The Principles and Practice of Electron Microscopy, Cambridge University press 2nd edn. (1997), (ISBN: 9780521435918). 87. P. Gabbott, Principles and Applications of Thermal Analysis, WileyBlackwell publishing, USA (2007), (ISBN: 978-1-4051-3171-1). 88. M. S. Niasaria, N. Mir, and F. Davar, Polyhedron 28, 1111 (2009). 89. L. D. Gelb and K. E. Gubbins, Langmuir 14, 2097 (1998). 90. K. H. Jones and J. A. Senft, J. Histochem. Cytochem. 33, 77 (1985). 91. G. Brunius, Current Microbiol. 4, 321 (1980). 92. T. Decker and M. L. Lohmann-Matthes, J. Immunol. Meth. 115, 61 (1988). 93. C. Korzeniewski and D. M. Callewaert, J. Immunol. Meth. 64, 313 (1983). 94. A. Vanka, S. K. Vishwakarma, G. Shanthi, K. B. Manohar, and A. A. Khan, IJAR 2, 561 (2014). 95. M. V. Berridge and A. S. Tan, Archives Biochem. Biophys. 303, 474 (1993). 96. A. A. Khan, A. K. Kooper, N. Parveen, S. Shakeel, et al., Indian Journal of Gastroenterology 2 (2002).

13