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Graphene and Ionic Liquid Matrices for Pathogenic Bacteria & Metallodrugs Analysis and Biosensing
Applications
Hani Nasser AbdelhamidMay 07-2013
5. Vacuum Pump
Extracting Lens
Accelerating Lens
MALDI Plume
Detector
Analyte
MALDI Target
Det
ecto
r
Detector
Refractron
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
Bacteria
• 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
Colorimetric Assay SPME coupled with GC-MS
FISH
Metallodrug applications
Anticancer “ Chemotherapy” Analytical Chemistry
Application No.1
MALDI analysis of Metallodrug
DHB Sinapinic acid
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
NH
OH O
F
FF
Cu+2
pH = 7.4
25.0 °C
Cu
NH
O O
F
FF
NH
OO
F
F F
H2O
GALDI-MS
Cu
[Cu(FF)2(H2O)2+H]+
m/z
= 6
61.0
Scheme that shows functionalization graphene nanosheet via noncovalent bond to assist noncovalent bondings between metals and drugs for GALDI-MS.
290 300 310 320 330 340 350 360 370 380 390 4000
2
4
6
8A
bso
rptio
n(a
r.in
t)
Wavelength, nm
Graphene
4000 3500 3000 2500 2000 1500 1000 500
Tra
nsm
issi
on
%
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
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.
250 260 270 280 290 3000
2
4
6
8
10
Abs
orpt
ion
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
d2 λ/
dλ2
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
d7 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).
280 300 320 340 360 380 4000.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5A
bsor
ptio
n(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
Abs
orp
tion(
Ar.
Val
ue)
Wavelength, nm
Fulfenamic drug Fe(II)-Fulfenamic complex
300 320 340 360 380 4000
2
4
6
8
10
Abs
orp
tion
Wavelength, nm
Fulfenamic acid Fe(III)-Fulfenamic complex
AB
C
Fig. 5. MALDI-MS spectra of Pseudomonas aeruginosa in positive mode at t = 10 min (A) and 12 h (B).
Fig. 5. MALDI-MS spectra of Pseudomonas aeruginosa in positive mode at t = 10 min (A) and 12 h (B).
Optical density of the bacteria with the parent drug and its complexes reported at 3 h (E) and 12 h (F).
400 450 500 550 6000
3
6
9
12
15
18
21
24
27
30
Flo
ure
sce
nce
In
ten
sity
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.
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).
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).
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.
Application No.2
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
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
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
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.
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.
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.
Table 1Physical parameters of conventional matrix 2,5-DHB and sinapinic acid (SA) and their related ionic liquid matrices.
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.
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).
Ab
solu
te I
nte
nsi
ty
(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.
Ab
solu
te I
nten
sity
(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.
Conclusion
Improve pathogenic bacteria.Low or no interference.
Improve physical and stability of conventional matrices
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.
A person who never made a mistake never tried anything new. Albert Einstein
Please, Feel Free to ask your question
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