This document describes the development of a cholesterol biosensor based on immobilizing cholesterol oxidase (ChOx) enzyme onto cobalt oxide (CoOx) nanoparticles. Cobalt nanoparticles were uniformly electrodeposited onto a glassy carbon electrode to form a porous nanostructure. ChOx was then immobilized via repetitive potential cycling. The biosensor detects cholesterol by measuring the hydrogen peroxide produced during the ChOx enzymatic reaction with cholesterol. Characterization with SEM, XRD, and AFM showed uniform CoOx distribution and enzyme immobilization. Cyclic voltammetry demonstrated the CoOx nanoparticles effectively mediate the electrochemical oxidation of hydrogen peroxide without needing an additional electron transfer mediator. The biosensor utilizes the Co

1. NANOSTRUCTURED
METAL OXIDE-BASED BIOSENSORS

2. BIOSENSOR
It is based on a biochemical reaction that occurs between an analyte and a biocatalyst
Immobilized on a suitable substrate.
Interaction between biocatalyst and a biomolecule is effected by
• Surface area, surface charge, energy, roughness
• And porosity, valence/conductance states, functional groups, physical
• States and hygroscopic nature all affect the formation of a biointerface.

3. NPG ASIA MATERIALS | VOL 3 | JANUARY 2011 | www.natureasia.com/asia-materials

5. The enzymatic reaction can be electrochemically monitored either by the decreased oxygen content in the solution or detection
formed hydrogen peroxide
The main immobilizing approaches adopted for cholesterol biosensor
• Self assembled monolayers
• Lipid bilayer memberane
• Anodic porous alumina
• Graphite-Teflon composite
• Prussian blue/poly pyrrole composite
• Direct adsorption Chitosan film
• Sol-gel composite film
Metal oxide matrix for enzyme immobilization has become attractive due to
• high ratio surface area
• uniform open pore structure
• chemical and thermal stability

6. Cobalt nanoparticles possess many good properties such as
• Low cost
• Biocompatibility
• Uniform film-forming ability
• No toxicity
Immobilization of ChOx onto a GC Electrode
Repetitive potential cycling (30 cycles at 100 mV s1 at potential range between 1.2 and1.1 V) in phosphate
buffer solution pH 7, containing 1 mM cobalt-chloride was used for electrodeposition of the cobalt oxihydroxide film
on the surface of the GC electrode.
{Electrophoretic deposition is that colloidal particles suspended in a liquid medium migrate under the influence of an electric
field (electrophoresis) and are deposited onto an electrode.}
After the deposition of cobalt oxihydroxide nanoparticles onto GC electrode, the electrode was immersed in fresh phosphate
solution containing 5 mg mL1 ChOx and the potential was repetitively cycled (20 scans) from 1.2 to
1.0 Vat scan rate 50 mV s1 for immobilization of ChOx.
Finally, the modified electrode was removed from ChOx solution, washed with double distilled water and stored at
refrigerator (4 °C) before being used in experiments

7. SEM image
Electrodeposited film exhibits uniform and porous cobalt-oxide
nanostructure containing nanowires with average diameter
ranging from 40 to 70 nm.
XRD image
No peaks corresponding to cobalt oxide were observed.
AFM images
• CoOx distributed uniformly on the electrode surface
• After immobilization of Cholesterol oxidase enzyme,
the surface morphology was changed and AFM image, with more
aggregated appearance was observed .
Voltammetric Response of the Biosensor to Cholesterol
• Determination of H2O2 which produced during enzymatic
oxidation of cholesterol is a strategy for cholesterol detection.
• We confirmed that CoOx nanoparticles are excellent electron transfer mediator
for H2O2 oxidation

8. Result and discussion
Characterizations of CoOx Nanomaterials
• The cyclic voltammogram, SEM image and XRD spectra
• The electrodeposited CoOx nanoparticles were taken
Cyclic Voltameter
• Formation of CoOx layer on the surface of electrode was checked by recording
cyclic voltammograms of the modified electrode in alkaline solution (pH 12) without
cobalt ions.
• Three oxidation peaks were observed at 0.1, 0.22 and 0.55 V during the
positive potential scan.
• These results can be attributed to the conversion between four different
cobalt oxidation phases of Co(OH)2, Co3O4, CoOOH and CoO2 which are stable
at alkaline pH.
• During the cathodic scan, two reduction peaks at 0.54 and 0.2 V were
observed, corresponding to the reduction of the various CoOx species formed
during the positive sweep.

12. Conclusion
• Cholesterol biosensor was made based on immobilizing ChOx onto CoOx nanoparticles without adding
any electron transfer mediator.
• The experimental results show that the ChOx enzyme remains active upon immobilization onto CoOx nanostructures.
• Determination of cholesterol was done by detection of generated hydrogen peroxide in the enzymatic reaction.