1. Graphene and Ionic Liquid Matrices for Pathogenic
Bacteria & Metallodrugs Analysis and Biosensing
Applications
Hani Nasser Abdelhamid
May 07-2013

2. 5. Vacuum Pump

3. Extracting Lens
Accelerating Lens
MALDI Plume
Detector
Analyte
MALDI Target
Detector
Detector
Refractron

5. COOH
OH
OH
H3CO
OH
OCH3
COOH
OH
COOH
CN
2,5-dihydroxy benzoic acid
3,5-dimethoxy-4-hydroxycinnamic acid
α-Cyano-4-hydroxycinnamic acid

7. Bacteria

8. • Polymerase Chain Reaction
• Culture and colony counting
methods
• Genosensor
• ELISA
• Amperomric Methods.
• Potentiometric Methods
• Electrochemical Impedance
Spectroscopy (EIS)
• Fluorescence Detection
• Surface Plasmon Resonance
• Piezoelectric Biosensor

9. Colorimetric Assay
 SPME coupled with GC-MS
FISH

10. Metallodrug applications
Anticancer “ Chemotherapy” Analytical Chemistry

11. Application No.1

12. MALDI analysis of Metallodrug
DHB Sinapinic acid

14. The 2010 Nobel Prize in Physics has been awarded jointly to Andre Geim and Konstantin
Novoselov "for groundbreaking experiments regarding the two-dimensional material graphene".
Andre Geim
Both physicists work at the University of Manchester in the UK.
 in 1859 Benjamin C. Brodie - potassium chlorate and fuming nitric acid.
In 1957 Hummers and Offeman - sulfuric acid H2SO4, sodium nitrate NaNO3, and
potassium permanganate KMnO4, which is still widely used (as of 2009).
1859, 1957, 2004
History

15. NH
OH O
F
F
F
Cu+2
pH = 7.4
25.0 °C
Cu
NH
O O
F
F
F
NH
O
O
F
F F
H2O
GALDI-MS
Cu
[Cu(FF)2(H2O)2+H]+
m/z=661.0
Scheme that shows
functionalization
graphene nanosheet via
noncovalent bond to
assist noncovalent
bondings between
metals and drugs for
GALDI-MS.

16. 290 300 310 320 330 340 350 360 370 380 390 400
0
2
4
6
8
Absorption(ar.int)
Wavelength, nm
Graphene
4000 3500 3000 2500 2000 1500 1000 500
Transmission%
Wavenumber Cm
-1
Graphene nanosheetC D
Characterization of graphene by using various instruments (A) UV, (B) TEM, (C) SEM
and (D) FT IR.
A B

17. Compound/complex pH Conductivity (S.Cm-1
)
Fulfenamic 4.11 150.0
Cu(II)-Fulfenamic complex 3.81 161.4
Fe(II)-Fulfenamic complex 3.91 162.6
Fe(III)-Fulfenamic complex 2.78 224.3
Table S1: pH and conductivity of fulfenamic drug and its complexes.

20. 250 260 270 280 290 300
0
2
4
6
8
10
Absorption
Wavelength, nm
Fulfenamic acid without graphene
Fulfenamic acid assisted with graphene
200 210 220 230 240 250 260 270 280 290 300 310 320 330
-6
-4
-2
0
2
4
6
8
d
2
λ/dλ
2
Wavelength, nm
Fulfenamic gas
fulfenamic assisted in graphene
200 210 220 230 240 250 260 270 280 290 300 310 320 330
-4
-3
-2
-1
0
1
2
3
4
d
7
λ/dλ
7
Wavelength, nm
Fulfenamic acid gas
Fulfenamic acid assisted by graphene
UV spectra of fulfenamic acid in gas phase with and
without graphene using first derivative (B),
second derivative (C) and seventh derivative (D).

21. 280 300 320 340 360 380 400
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Absorption(ar.int)
Wavelength, nm
Fulfenamic acid
Cu(II)-Fulfenamic complex
280 300 320 340 360 380 400
0.0
0.5
1.0
1.5
2.0
2.5
3.0
Absorption(Ar.Value)
Wavelength, nm
Fulfenamic drug
Fe(II)-Fulfenamic complex
300 320 340 360 380 400
0
2
4
6
8
10
Absorption
Wavelength, nm
Fulfenamic acid
Fe(III)-Fulfenamic complex
A
B
C

22. Fig. 5. MALDI-MS spectra of Pseudomonas aeruginosa in positive mode at t =
10 min (A) and 12 h (B).

23. Fig. 5. MALDI-MS spectra of Pseudomonas aeruginosa in positive mode at t =
10 min (A) and 12 h (B).

24. Optical density of the bacteria with the parent drug and its complexes reported at 3 h
(E) and 12 h (F).

25. 400 450 500 550 600
0
3
6
9
12
15
18
21
24
27
30
FlourescenceIntensity
Wavelength, nm
Fulfenamic acid
Bacteria(Pseudomonas aeroginosa, Staphylococcus aureus)
Fulfenamic acid + Bacteria
Emission spectra of pathogenic bacteria (Pseudomonas aeruginosa, Staphylococcus
aureus) and flufenamic acid at excitation wavelength of 360 nm.

26. Fig. 6. Fluorescence spectra of flufenamic acid and its complexes with Pseudomonas aeruginosa at ex = 360 nm. (A)
flufenamic acid (B) [Cu(II)(FF)2(H2O)2], © [Fe(II)(FF)2(H2O)2], (D) [Fe(III)(FF)3(H2O)2]. The inset show the linear
relationship between the difference of fluorescence intensity with the different colony of bacteria (cfu mL−1).

27. Fig. 6. Fluorescence spectra of flufenamic acid and its complexes with Pseudomonas aeruginosa at ex = 360 nm. (A)
flufenamic acid (B) [Cu(II)(FF)2(H2O)2], © [Fe(II)(FF)2(H2O)2], (D) [Fe(III)(FF)3(H2O)2]. The inset show the linear
relationship between the difference of fluorescence intensity with the different colony of bacteria (cfu mL−1).

28. Limit of detection(cfu/mL) Linear Range R2
Fulfenamic acid 3.4x104
2.0x104
– 4.5x104
0.87356
[Cu(FF)3(H2O)2] 3.4x103
2.0x103
- 4.0x103
0.99112
[Fe(FF)2(H2O)2] 3.3x103 2.0 x 103
– 6.5 x103
0.98323
[Fe(FF)3(H2O)2] 5.0x103
2.0 x103
- 5.0x103
0.9816
LOD (cfu/mL) Linear range R2
Fulfenamic acid 4.9x104
2.1 x 104
– 5.0 x104
0.99475
[Cu(FF)3(H2O)2] 3.4x103
2.3 x 103
– 5.5 x 103
0.98743
[Fe(FF)2(H2O)2] 3.9x103
2.2 x 103
– 5.0x 103
0.98112
[Fe(FF)3(H2O)2] 4.5x103
2.5x103
– 5.5x103
0.9956
Table S7: Limit of detection of Staphylococcus aureus with parent drug and its complexes.
Table S6: Limit of detection of Pseudomonas aeruginosa with parent drug and its
complexes.

29. Application No.2

30. OH
COO
-
OH
NH3
+
OH
COO
-
OH
NH
+
CH3
CH3
a) DHB/ANI b) DHB/ DMANI
OH
COO
-
OH
OH
COO
-
OH
c) DHB/DCHA
NH2
+
NH2
+
CH3
CH3
d) DHB/DMA
OH
COO
-
OH
OH
COO
-
OH
e) DHB/py
NH
+
NH
+
CH3
f) DHB/2P
OH
COO
-
OH
NH
+
CH3
g) DHB/3P

31. OH
H3CO
H3CO
COO
-
NH3
+
OH
H3CO
H3CO
COO
-
NH
+
CH3
CH3
a) SA/ANI b) SA/ DMANI
OH
H3CO
H3CO
COO
-
OH
H3CO
H3CO
COO
-
c) SA/DCHA
NH2
+
NH2
+
CH3
CH3
d) SA/DMA
OH
H3CO
H3CO
COO
-
OH
H3CO
H3CO
COO
-
e) SA/py
NH
+ NH
+
CH3
f) SA/2P
OH
H3CO
H3CO
COO
-
NH
+
CH3
g) SA/3P

32. Matrix m/z Assignments
SA/ANI
316.6 [M+H]+
SA/DMANI
345.8 [M+H]+
SA/DCHA
405.6 [M+H]+
SA/Pyr 303.8 [M+H]+
SA/2-P
317 [M+H]+
SA/3-P
317 [M+H]+
SA/DEA
299.9 [M+H]+
Matrix m/z Assignments
2,5-DHB/ANI
248.0 [M+H]+
2,5-DHB /DMANI
275.8 [M+H]+
5-DHB /DCHA
335.6 [M+H]+
2,5-DHB /Pyr
332.8 [M+H]+
2,5-DHB /2-P
248.2 [M+H]+
2,5-DHB /DEA
228.7 [M+H]+
Table: ESI spectra of Ionic liquid matrices

33. Fig. 3. UV spectrum of (a) conventional matrix (a) SA, (b) 2,5-DHB and its ionic
liquid matrixes. The vertical line represents wavelength of laser used in UV-
MALDI-MS.

34. Fig. 1. MALDI-MS spectrum of pseudomonas aeruginosa using 2,5-DHB and ionic
liquid matrices, (a) 2,5-DHB, (b) 2,5-DHB/ANI, (c) 2,5-DHB/DMANI, and (d) 2,5-
DHB/pyr.

35. Fig. 2. MALDI-MS spectrum of Pseudomonas aeruginosa using SA and ionic liquid
matrices, (a) SA, (b) SA/ANI, (c) SA/DMANI, (d) SA/DCHA (e) SA/Pyr, (f) SA/2-P, (g)
SA/3-P and (h) SA/DEA.

36. Table 1
Physical parameters of conventional matrix 2,5-DHB and sinapinic acid (SA) and their
related ionic liquid matrices.

37. Fig. 4. Schematic representation of MALDI-MS of conventional and ionic liquid matrices. Conventional matrix, are weak acids so it show
low proton exchange. In other side, hydrogen bond in ILs promote proton exchange between the matrices and bacteria.

38. Fig. 5. MALDI-MS spectrum of Staphylococcus aureus using (a) conventional matrixes SA, (b) aliphatic ionic liquid matrixes AILM
(SA/DCHA, SA/DEA respectively), (c) aromatic ionic liquid matrixes ARILM (SA/ANI, SA/DMANI respectively) and (d) heterocyclic
ionic liquid matrixes HILM (SA/Pyr, SA/2-P, SA/3-P respectively).

39. AbsoluteIntensity
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Fig. S7: Interference peaks of 2,5-DHB series. (a) 2,5-DHB matrix (b) 2,5-DHB/ANI, (c) 2,5-DHB/DMANI (d) 2,5-DHB/DCHA, (e) 2,5-
DHB/Pyr, (f) 2,5-DHB/2-P,(g) 2,5-DHB/3-P,(h) 2,5-DHB/DEA.

40. AbsoluteIntensity
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Fig. S8: Interference peaks of SA series (a) SA matrix (b) SA/ANI, (c) SA/DMANI (d)
SA/DCHA, (e) SA/Pyr, (f) SA/2-P,(g) SA/3-P,(h) SA/DEA.

42. Conclusion
 Improve pathogenic bacteria.
Low or no interference.
Improve physical and stability of
conventional matrices

43. Acknowledge
* Assuit university, Egypt
* National sun-yat sen university (NSYSU), ROC.
* Prof. H.-F.Wu.
* Prof. Shiea *Prof. jiang.
* Prof. Tseng. *Prof. Yang Hsiang Chan
*My colleagues and My lab mate.

44. A person who never made a
mistake never tried anything new.
Albert Einstein
Please, Feel Free to ask your question