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

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