This document summarizes a research article that presents a novel biosensor for detecting nitrite based on a single-layer graphene nanoplatelet (SLGnP)-protein composite film. The composite film is created by combining SLGnP, tetrasodium 1,3,6,8-pyrenesulfonic acid (TPA), and myoglobin protein and casting it on a glassy carbon electrode. Characterization shows the protein retains its structure and there are strong electrostatic interactions between the components in the film. Electrochemical analysis demonstrates the composite film enhances the direct electron transfer of the protein and can catalytically reduce nitric oxide. The resulting biosensor exhibits high sensitivity, a low detection limit, good stability and reproducibility for

1. A Novel Nitrite biosensor based
on Single-layer Graphene
nanoplatelet(SLGnP)-protein
composite film
12-06-2011
Rong Yue, Qing Lu and Yikai Zhou*
Biosensors and Bioelectronics 26(July 2011) 4436-4441
Tejasvi Parupudi

2. Yikai Zhou*
Rong Yue
Qing Lu

3. Lead PI Prof. Yikai Zhou
Affiliation MOE Key Lab of Environment and Health
School of Public Health,
Tongji Medical College
Huazhong University of Science and Technology,
Wuhan 430030, PR China
Co-Authors Prof. Rong Yue
Prof. Qing Lu
About the Authors

4. Prof. Yi-Kai Zhou
• Director of Environment and health key laboratory
under Ministry of Education (MOE)
• Director of Environmental protection and health key
laboratory under Environmental protection bureau
• Research Interests: Composite films for biosensing,
nanocomposite film properties, development of
analytical systems for biological component detection,
cyclic voltammetry based studies
• Specific interest in current paper: nanoplatelet film
composition, properties and analysis using CV, XPS
• Affiliation: MOE Key Lab of Environment and Health,
School of Public Health, Tongji Medical College

5. Prof. Qing Lu
• Research Interests: Cyclic Voltammetry based
studies of composite films, characterization of
composite films for interesting properties using
scanning probe microscopy
• Specific interest in current paper: Nitrite
sensitivity to composite films, graphene films
• Affiliation: School of Materials Science and
Engineering, Nanyang Technological University,
Singapore 639798, Singapore and MOE Key Lab,
Tongji Medical College, HUST, China.

6. Prof. Rong Yue
• Research Interests: Amperometric sensors,
electrochemical studies of composite films
• Specific interest in current paper: composite
film characteristics, amperometric sensing
• Affiliation: MOE Key Lab of Environment
and Health, Tongji Medical College, HUST,
China.

7. Useful terms
• Supernatant- liquid remains above the solid after gravity
sedimentation and centrifugation(SLGnP-TPA)
• Nanoplatelet- nanoscale Graphene monolayer
• Isoelectric Point- pH at which a protein carries no net charge
• Nafion- a synthetic polymer with ionic properties used as a
binder to hold the composite film onto the electrode
• TPA- tetrasodium 1,3,6,8-pyrenesulfanoic acid – an aromatic
molecule used to exfoliate graphite into graphene monolayers

8. Motivation
Nitrite detection- Why so important?
• Preservatives- processed meat- curing
• Fertilizing agents
• Carcinogenic features- nitrosamines
• Principal Reaction:
Need of the hour:
• Fast and in situ monitoring of nitrite

9. Blue Baby Syndrome
Ref: By Author: Jules Atkins, RM Supplied by: Brandi Catt (Transferred from
en.wikipedia), via Wikimedia Commons

10. Role of Graphene
• Nontoxicity
• Chemical, thermal tolerance
• Electric conductivity
• Mechanical hardness
• Electric sensitivity to perturbations
• Large surface area (useful in catalysis)

11. Existing well-known detection
schemes/techniques
• Spectrophotometry(Griess Reaction)
• Ionic Chromatography Polarography
• Capillary Electrophoresis
• Fluorescence Spectrophotometry
• Enzymatic biosensors(Nitrite Reductase NiR)
In simple words, Griess assay, fluorescent assays, chemiluminiscence assays and
electrochemical detection

12. Properties of SLGnP
Single-layer Graphene nanoplatelet film
• High electric sensitivity
• Biocompatible
• High conductivity
• Large effective surface area
• Electrocatalytic reduction capability
• Suitable electron-transfer distance
• Useful in direct electrochemistry-based biosensors

13. Reagents : I. Myoglobin (Mb)
F. G. Parak and G. U. Nienhaus, Myoglobin- a paradigm in the study of protein
dynamics, ChemPhysChem 2002, 3, 249 ― 254
A Heme-protein responsible for carrying oxygen in mammals

14. II. TPA
Dong et al., Symmetry breaking of Graphene monolayers by molecular decoration Phys.
Rev. Lett. 102, 135501 (2009)

15. Techniques/Methods and
Background Fundamentals
• Preparation of composite film modified electrode
• Freshly prepared GC electrode
• Mb dissolved in tri-distilled water added to SLGnP-TPA
in 1:1 ratio
• Nafion added to SLGnP-TPA-Mb film modified electrode
to enable binding of film to electrode
• Final electrode representation:
Nafion/SLGnP-TPA-Mb/GC electrode
Composite film

16. Results
I. Characteristics of the SLGnP-TPA-Mb composite film
Figure 1. FESEM images showing surface morphologies of A)Mb film (flat, apertured) and
B)SLGnP-TPA film(aggregated acicular narrow and long pointed crystal clusters)

17. Figure 1. FESEM images showing surface morphologies of C)SLGnP-TPA-Mb composite
film (homogenous, flake-like configuration) and D)SLGnP-TPA-Mb magnified image
(petal structures with widths of 200nm)
Conclusion: Strong interactions exist between the protein(Mb) and SLGnP-TPA
Mb: positively charged
SLGnP-TPA : negatively charged
Electrostatic attraction

18. SI-1. XPS spectra of Mb (A, B), SLGnP-TPA (C, D)
and SLGnP-TPA-Mb (E, F)
Comment:
A,B (protein Mb) and
E,F (composite film) show
N 1s peaks in their XPS spectra
protein could attach to composite
film due to electrostatic interactions
Mb retains its native structure within
the film environment

19. SI-2. XPS data analysis of Mb, SLGnP-TPA and
SLGnP-TPA-Mb
sample
C 1s O 1s N 1s S 2p Fe 2p
BE (eV) Atom %
BE
(eV)
Atom % BE (eV) Atom % BE (eV) Atom % BE (eV)
Atom
%
Blank 284.63 21.23 531.98 77.49 399.33 1.28
Mb 284.61 69.44 531.03 18.6 399.33 10.74 168.15 1.08 706.25 0.13
SLGnP-TPA 284.6 47.5 531.71 44.25 401.33 0.32 168.11 7.93
SLGnP-TPA/Mb 284.64 64.48 530.9 23.36 399.36 9.53 167.77 2.47 706.06 0.15

20. UV-Vis ABSORPTION spectra of a)SLGnP-
TPA-Mb b)Mb and c)SLGnP-TPA solutions
355 nm
476 nm
UV absorption of TPA
Comment: The protein (Mb) can retain its natural structure in the composite

21. II. Electrochemical characteristics of the SLGnP-TPA-Mb composite film
Comment: Epa=-0.385V ; Epc=-0.419V ; ΔEp= 34 mV
=> better direct electron transfer enhancement through SLGnP(GRAPHENE)
Figure 3. Cyclic voltammograms at 0.1 V s-1 in pH 7.4 buffers for (a) Nafion/SLGnP–TPA–Mb
film (dark), (b) Nafion/SLGnP–TPA film (dark yellow), (c) the bare GC electrode (blue), (d)
Nafion/Mb film (magenta) and (e) Nafion/TPA–Mb film (red).

22. Effect of scan rate on electrochemical response of
composite film by CV- Speed test!
Figure 4: Cyclic voltammograms at SCAN RATE: 0.1 V s−1 in pH 7.4 buffers for Nafion/SLGnP–TPA–
Mb film at scan rates of (a) 0.5, (b) 0.4, (c) 0.3, (d) 0.2, (e) 0.1, (f) 0.08, (g) 0.06 V s−1.
The inset are plots of the reduction and oxidation peak current “i” against the scan rate “v” (upper) and
the logarithm of “i” against the logarithm of “v” (lower).
Comment: SLGnP-TPA is an excellent promoter for electron transfer between the protein (Mb) and
the electrode (GC) ; Rate constant: 3.9 s-1

23. Effect of phosphate buffer pH on electrochemical
response of composite film by CV
Figure 5: Cyclic voltammograms at 0.1 V s−1 for Nafion/SLGnP–TPA–Mb film in phosphate buffer solutions
of pH (d) 3.0, (e) 5.0, (f) 7.4, (g) 9.0. The inset shows the relationship between (a) Epc, (b) Epa, (c) E◦ of
Nafion/SLGnP–TPA–Mb film and the solution pH value.
Comment: Asymmetric redox CV is due to a reversible pH-induced conformational change of Mb
at low pH

24. Figure 6: Cyclic voltammograms at 0.1 V s−1 in pH 5.0 buffer for (a) Nafion/SLGnP–TPA and
(c) Nafion/SLGnP–TPA–Mb films in buffer without NaNO2,(redox peaks at -0.4V) (b) Nafion/SLGnP–
TPA and (d) Nafion/SLGnP–TPA–Mb films in buffer containing 0.3 mM NaNO2(cathodic peak at -0.77 V)
(e) Nafion/SLGnP–TPA–Mb film in buffer containing 0.7 mM NaNO2. The inset is the plot of catalytic peak
current vs the concentration of NaNO2.
III. Electrocatalytic properties of the SLGnP-TPA-Mb composite film
Comment: Cathodic peak appeared due to reduction of nitric oxide catalysed by Mb

25. Electrocatalytic reduction of nitric
oxide at the Nafion/SLGnP–TPA–Mb film
• MbFe(III) + e → MbFe (II)
• MbFe(II)+NO→MbFe (II)–NO
*
• MbFe(II)–NO+2e+2H+→MbFe(II)+H2O+N2O
Some intermediate reactions:
*Bayachou, M., et al, 1998. J.Am.Chem.Soc. 120,9888-9893
Comment: Due to linear relation between cathodic peak current and conc. of NO2
-
Nafion/SLGnP-TPA/Mb film can be used as an amperometric sensor for nitrite detection

26. Features of the biosensor
• Sensitivity: 3.42 x 104 µAM-1cm-2
• Detection limit: 0.01mM at SNR 3(greater than
Hb-silk fibroin film and HB/CTAB)
• Stability: 200 continuous CV cycles between -
0.1 to 1.2 V in pH 5 buffer with 0.1Vs-1 scan rate
• Reproducibility: RSD of 4.1% for 8 successive
measurements
• No influence of major biological fluid
interference(uric acid 10 times more concentrated
than NO2
-)

27. Critique
• Graphene proved to be a valuable asset due to its amazing surface
properties
• The composite film reduced the electron transfer distance between
redox center of the heme protein(Mb) and the electrode surface
• The sensor can be developed into a commercial product due to its ease
of fabrication and excellent biocompatibility
• No special conditions needed; experimentation at room temperature
• The protein Mb retains its structure in the composite film, critical to the
functioning of the sensor
• A well-defined (optimal) pH is required for the interaction between the
protein and electrode
• Atomic Force Microscopy would have given a clearer picture of the film
morphology and various force tests on the film could be done
• It should be possible to design a new protein structure that has more
electroactive centers for interaction with the composite film to enable

28. Thank You
Questions?