It is a graphene interfaced electrical biosensor for for label free and sensitive detection of E.coli 0157:H7

1. GRAPHENE-INTERFACED ELECTRICAL BIOSENSOR FOR LABEL-FREE AND SENSITIVE
DETECTION OF FOODBORNE PATHOGEN E.coli O157:H7
PRESENTED BY
KAMARAPU MOUNIKA(NIPERA1618MD04)
ANUP KUMAR(NIPERA1618MDO1)
DEPT. OF MEDICAL DEVICES
GUIDED BY
Dr. Mukty sinha
Dr. Govind Kapusetti 1

2. ARTICLE INFORMATION
PUBLISHED BY:-
ELSEVIER
AUTHORS:-
Ashish Pandey, Yasar Gurbuz, Volkan
Ozguz, Javed H.Niazi, Anjum Qureshi
IMPACT FACTOR:-
7.47
DATE OF PUBLICATION:-
16 December,2016
JOURNAL:-
Biosensors and
Bioelectronics
2

3. TRACK OF SEMINER
INTRODUCTION
MATERIALS
&METHODS
RESULTS &
DISCUSSION
DRAWBACKS &
FUTURE
PERSPECTIVE
CONCLUSION
3

4. 4

5. BIOSENSOR
Biosensor is a self contained
integrated device that can able to
providing specific qualitative or semi-
quantitative analytical information
using a biological recognition element
which is in direct or spatial contact
with a physio-chemical transducer
element to produce a electrical signal
proportional to a single analyte which
is then conveyed to a detector
5

6. COMPONENTS OF BIOSENSOR
6

7. Escherichia coli O157:H7
 E.coli O157:H7 is a very harmful food borne
pathogen causing severe human disease like
hemolytic uremic syndrome, bloody diarrhea etc.
 It is a Gram- negative, rod-shaped microorganism
that expresses somatic (O) antigen 157 and
flagella(H)antigen 7.
 Shiga toxin and products of the F-like plasmid are
the major virulence factor
 The chromosomal size of E.coli O157:H7 is 5.5 MB
and the size of only F-like plasmid is 92-104 KB 7

8. CONVENTIONAL TECHNIQUE
E.coli
O157:H7
Detection
Polymerase
Chain
Reaction
DNA
Microarray
DNA
Sequencing
Technology
ELISA
Staining
8

9. CONVENTIONAL BIOSENSOR
Electrochemical immunosensor based on
graphene-wrapped copper oxide-cysteine
hybrid composite
Graphene based field effect transistor
biosensor
9

10. DRAWBACKS
The suspected samples have to be extensively processed for
pathogen detection , such as cell lysis, DNA extraction etc.
Require secondary chemical mediators such as fluorophores
Using of redox- mediators which hampers signal transduction
Low specificity and sensitivity due to cross-reactivity
Less accuracy
expensive
10

11. SiO2 WAFER
GRAPHENE
GOLD
MICRO
ELECTRODE
PASE LINKER
ANTI-E.coli E.Coli
O157:H7
111

12. MATERIALS PROCURED FROM
Graphene nanoplatelets Carbon Solution Inc., USA
Monolayered graphene Carbon Solution Inc., USA
N,N dimethylformamide Sigma-Aldrich
SiO2 - substrates Graphene platform corp., Japan
E.Coli O157:H7 NCTC 12900, Public Health England
12

13. WHY GRAPHENE CHOOSED
Its having both holes and pi(π) electrons as
charge carriers, located above and below of
the graphene sheet
Its having high charge-mobility at room
temperature with tunable conductance
Its having highly inherent strength due to
presence of 0.142 nm-long carbon bonds
Integrating graphene nanostructures
enhancing sensitivities of electrical sensor
Large surface area of graphene enables
homogeneous of biological materials
13

14. COUPLING OF GNPs & MG ON SiO2 -SUBSTRATES
GNPs were first suspended in DMF at a concentration of 0.5 mg/ml
and homogenized by ultrasonication for 5 h.
The colloidal GNPs suspension was stable for 6-7 h and this
suspension was subjected to spin coating on SiO2 wafer
Monolayered graphene coupled on SiO2 – substrates by chemical
vapor deposition technique
14

15. PATTERNING OF GOLD INTERDIGITATED
MICROELECTRODES ON SiO2 SUBSTRATES INTERFACED
WITH GNPs/MG & CHARACTERIZATION
Both type of graphene coupled on SiO2 substrates were subjected
to patterning the gold microelectrodes by using
photolithograpgy technique
The fabricated GNPs/MG interfaced with electrical chips were
characterized by SEM and Raman microscopy technique
15

16. ACTIVATION OF GRAPHENE-INTERFACED WITH
ELECTRICAL BIOCHIP & CHARACTERIZATION
GNPs /MG interfaced with capacitor arrays were incubated with 5 mM of 1-pyrenebutanoic acid succinimidyl
ester in methanol for 2 hour
A self assembly of PASE was formed on outermost layer of graphene on chip
Sensor were then washed with methanol followed by water and dried
PASE activated sensor chip were incubated overnight with anti E.coli O157:H7 antibodies (1,3 and 5 µg/ml in
PBS) at 4ºc
The chips were incubated with 5% BSA in PBS solution containing 0.1% Tween 20
Immobilization of anti E. coli O157:H7 on graphene interfaced with chip were confirmed by FT-IR
spectrometer and contact angle measurement by sessile drop method 16

17. BACTERIAL CULTURES AND INCUBATION OF
CELLS ON CHIP
A wild type non -specific E.coli strain ( DH5α) was used as a negative
control for validation
Replicate cell suspensions ranging from 10-107 cells/ml were prepared
freshly just before the biosensor processes
All cell concentration in 5 µl volumes were incubated on capacitor arrays
functionalized with anti- E. coli O157:H7 antibodies for 30 min
The chips were washed with PBS followed by quick wash with water and
dried
17

18. ELECTRICAL MEASUREMENTS
Dielectric parameters of GNPs/MG interfaced with chips before and after
incubation with E . Coli O157:H7 cells (10-107 cells/ml) was measured against
the applied frequency from 40Hz and 7 MHz using a karl süss PM-5 RF probe
station and Agilent 4294 A impedance analyzer
Specificity test were conducted with E. coli(DH5-α )at 102 and 104 cells/ml
Analysis of dispersion characteristic frequency and relaxation time dependent
upon the cells concentration was calculated using equation
fc =1/2∏τ
18

19. CHARACTERIZATION OF GNPs AND MG
INTERFACED WITH ELECTRICAL BIOSENSOR CHIPS
SEM images of GNPs and MG
in between the gold
microelectrodes of chips
confirmed the presence of a
few layers of GNPs and MG
distributed on sensor chip
surface
19

20. SEM(CONT’D)
GNPs appeared uniformly
distributed but provided
rough surfaces in between
the microelectrodes of
chips due to their multi
layers compared to MG on
SiO2 substrate
20

21. RAMAN SPECTROSCOPY
 Raman spectra of GNPs on chip
showed 2D<G , which indicate the
presence of a few layers of
graphene on sensor surface
 Raman spectra of MG on sensor
surface showed that 2D>G ,
indicating the presence of defect
free MG layer on chip
21

22. FUNCTIONALIZATION OF GNPs/MG
CHIPS ANTI E.coli O157:H7 ANTIBODIES
Pyrene tetra-rings in PASE having a honeycomb –like structure, which promoted stacking
on outermost surfaces of GNP and MG by ∏-∏ interaction
The other terminal of PASE molecules having free NHS-ester group which helps covalent
attaching with amino groups of antibodies specific to E.coli O157:H7
22

23. E.coli O157:H7 CAPTURING
Capturing of E.coli cells on sensor surface by E.coli O157:H7 specific antibodies was
confirmed by examining under optical microscope that shows clear cell dependent
attachment
23

24. DETECTION OF E.Coli O157:H7 ON
ELECTRICAL CHIPS
 The capacitance get changed as a function of different concentration of E.Coli O157:H7 cells on GNPs and
MG –interfaced sensor
 Changes in capacitance profiles from sensor chips captured with O157:H7 cells (0-107 cells/ml) were
measured against the applied AC frequency (40 Hz-7 MHz)
 An appropriate applied frequency of 6 MHz was selected based on the prominent dispersion peak found in
GNPs-chip
24

25. CONT’D
At lower frequencies( 0-2.5 MHz) cells seemed to
develop resistance probably due to cell
membrane insulation from an external field.
At higher frequencies(4-7 MHz) , the charges on
cells-on-chip allow to electric field
The shift in dispersion peak from characteristic
frequency of 6.2MHz(with only antibodies) to
5.82MHz upon captured E.coli O157:H7 cells also
indicated its origination from induced dipoles
one cell surface.
25

26. CONT’D
The relaxation time of E.Coli O157 cells on GNPs increased with
increasing cell density from 102 to 106 cells/ml indicating
effective mobility of the of the surface charges/ions (dipole
moments) with applied electric field
Increase in cell-density on chips provided larger volume on
sensor surface that probably lead to changes in
intermolecular/cellular motion due to anisotropic change with
applied AC frequency
26

27. SENSITIVITY
The capacitance developed by MG and GNPs
biosensors was 4 pF and 1pF respectively when
concentration get changed from 10 to 100 cells/ml.
The MG-Chip provided greater sensitivity(10 cells/ml)
where as minimum sensitivity of GNPs is 100 cells/ml.
The sensitivity of MG chip was associated with defect
free mono layered graphene nano structure that
provided smooth topography and easy access for
surface antibodies
27

28. EQUIVALENT ELECTRICAL CIRCUIT OF A
CELL
The antibody probe immobilization on graphene
and cells binding on graphene sensor surface
influence changes in the charge density
(dielectric changes) on microelectrodes through
polarization of cell-surface charges ,intracellular
bioactivity and dipoles induced by cellular
electro negativity that contribute to overall
changes in surface conductivity and capacitive
responses of sensors
28

29. SENSOR SPECIFICITY
No significant increase in capacitance response with a non-pathogenic E.Coli DH5α strain, indicating
high specificity
A small loss of capacitance occurred with GNPs chips due to heterogeneous surface
29

30. FUTURE PROSPECTIVE
It can be used to detect other highly pathogenic bacterial species
like staphylococcus aureus, vibrio cholerae , mycobacterium
tuberculosis etc.
Use of substantially equivalent inexpensive biomaterial instead
of gold
Increase of more accuracy
30

31. LIMITATION…
Results of FTIR spectra and contact angle measurement
No comparison graphical data provided with existing
graphene based electochemical biosensor
31

32. CONCLUSION
Introducing a fabricated graphene based label free electrical chip
platform could overcome challenges related to other existing
methods that mainly function on the principle of dielectric changes
It is a simple, rapid, highly sensitive and very specific technique for E.coli
O157:H7 detection
MG biosensor provide higher sensitivity than GNPs. However, both
sensing platform serve as powerful tools for detection of pathogen
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