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

Yikai Zhou*

Rong Yue

Qing Lu

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

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

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.

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.

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

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

Blue Baby Syndrome

Ref: By Author: Jules Atkins, RM Supplied by: Brandi Catt (Transferred from

en.wikipedia), via Wikimedia Commons

Role of Graphene

• Nontoxicity

• Chemical, thermal tolerance

• Electric conductivity

• Mechanical hardness

• Electric sensitivity to perturbations

• Large surface area (useful in catalysis)

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

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

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

II. TPA

Dong et al., Symmetry breaking of Graphene monolayers by molecular decoration Phys.

Rev. Lett. 102, 135501 (2009)

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

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)

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

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

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

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

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).

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

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

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

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

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-)

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

Thank You

Questions?