Synthesis of graphene-based nanomaterials with ...€¦ · Synthesis of graphene-based...

Synthesis of graphene-based nanomaterials with applications in

the electrochemical detection of biomolecules

Stela Pruneanu

INCDTIM-Cluj-Napoca

NANOGENTOOLS Spring School May 2019- Alessandria

TOPICS 1. Graphene synthesis 2. Electrochemical detection of dopamine 3. Electrochemical detection of cancer biomarker (8-OHdG) 4. Conclusions


Single layer of sp2 hybridized carbon atoms High mobility of charge carriers: 200.000 cm2V−1s−1 Surface area of a single graphene sheet is 2630 m2/g Graphene is resistant to attack by powerful acids and alkalis (hydrofluoric acid, ammonia)

1. Graphene synthesis


Au(x)/MgO- catalyst, where x = 1, 2 or 3 wt% Ag(x)/MgO-catalyst, where x = 1,2 or 3 wt% Pt(x)/MgO-catalyst, where x = 1,2 or 3 wt%

AuAg(x)/ MgO-catalyst, where x = 1:1 or 1.5:1.5 wt% AuPd (x)/ MgO-catalyst, where x = 1:1 or 1.5:1.5 wt% AuCu (x)/ MgO-catalyst, where x = 1:1 or 1.5:1.5 wt% AuPt (x)/ MgO-catalyst, where x = 1:1 or 1.5:1.5 wt% Methane (CH4)- carbon source 1000 oC- synthesis temperature (60 minutes) Purification in HCl (30 minutes) Drying - 120 oC (overnight)

A.R. Biris et al, Carbon, 50 (2012) 2252

A) Chemical Vapor Deposition (CVD)-bottom up


Time-lapsed HRTEM images of the growing graphene layers on Pt recorded in situ under exposure of 1.3 mbar iso-butene at 475 C (a: 10 min; b: 1 s; c: 1.2 min; and d: 3.2 min)

Schematic illustration of graphene layer growth on Pt particles of increasing size: (c and d) envelopment of Pt particles by graphene for particles greater than 6 nm in diameter; (e and f) formation of graphene nanotubes on Pt particles of 2–6 nm; (g and h) formation of graphene sheets and their migration to the support for Pt particles less than 2 nm in diameter.

A.T. Bell et al., Journal of Catalysis 286 (2012) 22–29

iso-butene


http://www.google.ro/url?sa=i&source=images&cd=&docid=k9m5xcvmx1knoM&tbnid=CVzl4F8T37ttFM:&ved=0CAgQjRwwAA&url=http://commons.wikimedia.org/wiki/File:Isobutene_struttura.PNG&ei=eBVAUonrIKLG0QXan4HABA&psig=AFQjCNGg0ukpFrn1XUo9SFQ5j0GqrAus2Q&ust=1380017912670466


EDX analysis

F. Pogacean et al., International Journal of Nanomedicine 2014:9 1111–1125

TEM images of Graphene-AuAg

Au: Ag (1:1 wt.%) Au:Ag (1.5:1.5 wt.%)

AuAg: 5- 50 nm; 13 nm AuAg: 5-100 nm; 20 nm

Rate of electron transfer at the edge planes >> than that at the basal planes

C. E. Banks et al., RSC Adv., 2011, 1, 978–988.

HR-TEM images of Graphene-AuAg


Graphene flakes

Advantages of CVD method:

- Low amount of oxygen-containing groups (due to the lack of oxygen from the reaction chamber)

- The honeycomb structure is preserved

- Metallic nanoparticles are attached during synthesis (no need for additional reaction)

Disadvantages:

- Low quantity of graphene can be obtained after one synthesis (50 mg)


B) Chemical synthesis (top-down)

C. Socaci et al., Sensors and Actuators B 213 (2015) 474–483

Thermal reduction (300o Ar) Chemical reduction


Graphene-metallic nanoparticles (chemical)


Graphene/Au (10 – 40 nm)

Graphene/AuPd (10 – 30 nm)

Advantages of chemical synthesis:

- Large quantity of graphene-oxide (GO) can be obtained after one synthesis (8 g)

- After reduction - RGO (4 g)

Disadvantages:

- Chemical/Thermal treatment is needed, in order to reduce graphene-oxide

- Large amount of oxygen-containing groups (the honeycomb structure is not well preserved)


Dopamine- is a neurotransmitter and a hormone Concentration in body fluid : 1-10 µM Low levels of dopamine - Parkinson's disease Higher dopamine concentrations: schizophrenia, attention deficit hyperactivity disorder (ADHD), and restless legs syndrome (RLS).


2. Electrochemical detection of dopamine

Increases the active surface area (20-70 %) Current is proportional with surface

area Improves the transfer of electrons

Graphene modified electrodes for the detection of dopamine


CE WE RE

Screen-printed electrode GC or Au electrode, modified with graphene (10 microL)

Graphene suspension in DMF (1 mg/mL)

Identification of the FTIR absorption bands: 1716 cm-1 - C=O stretch of the COOH group 1125 and 1205 cm-1 -C-O stretching vibration of epoxy and alkoxy groups 1464 and 1570 cm-1 - OH deformation vibrations 3434 cm-1 - O-H stretch from the adsorbed H2O molecules

Graphene (CVD) vs Graphene (Chemical)


TEM images of graphene FTIR spectra of graphene

Graphene- CVD

Graphene- Chemical

Graphene- CVD

Graphene- Chemical

S. Pruneanu et al., Electrochimica Acta 154 (2015) 197–204


Electrochemical oxidation of dopamine

In acidic solution: -dopamine is present in the cationic form -pKa1 = 8.9 for aminoethyl group -pKa2 = 10.6 hydroxyl group - oxidation occurs as 2e/2H+ process, forming o-dopaminoquinone (o-DQ)

In neutral and basic solutions: -a cyclization reaction occurs, yielding leucodopaminochrome (LDC) - LDC undergoes a 2e/2H+ process, but at lower potentials


Graphene CVD- excellent electro-catalytic activity: The decrease of the peak potential ̴ 140 mV

Increase of the peak current ̴ 5 times




Au electrodes modified with graphene

𝐼𝐶 = 𝑑𝑄

𝑑𝑡= 𝐶

𝑑𝐸

𝑑𝑡; C = Q/V

Graphene-Chemical

Graphene- CVD

Ic = 5 x 10-5 A

Ic = 1 x 10-5 A




LOD = 10-7 M (graphene-CVD) Linear range: 3 x 10-7 – 10-4 M Covers well the concentrations in body fluid (10-6– 10-5 M)

LOD = 3 x 10-6 M (graphene –chemical) Linear range: 10-5 – 10-4 M Does not cover the concentrations in body fluid




Interfering species: ascorbic and uric acid Graphene-CVD



Ascorbic acid- an organic acid with antioxidant properties In the cells, it protect the biological molecules from oxidative degradation; stimulates the production of collagen, neurotransmitters or steroid hormones. Uric acid – an organic acid which results after degradation of purine bases of DNA (adenine; guanine) or RNA.

Interfering species: ascorbic and uric acid Graphene-CVD


Conclusions

- Graphene – CVD has better electrochemical properties comparing with Graphene-chemical

- The linear range and LOD obtained for graphene-CVD: fit well with the concentrations of dopamine in body fluid

- Ascorbic acid and uric acid influence the detection of dopamine


C) Electrochemical synthesis of graphene by exfoliation of graphite rods

Experimental set-up: - DC Power Supply (up to 30 V) - Two graphite rods (high purity; 99.995%) - Reaction vessel -Mixture of strong acids (sulfuric + nitric) - few hours synthesis time - wash, filtrate , centrifugation and dry


5 10 15 20 25 30 35

0

10

20

30

40

50

60

70

80

90

100

Measured

GO

FLG

MLG

Inte

ns

ity

(a

.u.)

2degrees)

11.9

5

22.8

9

26.0

4

11.95 22.89 26.04

No. of layers 3 4 14

% 55 23 2221L

GO GR

GO

GR

GO d spacing = 0.75 nm (insulating; good biocompatibility with living systems) GR d spacing = 0.36 nm (highly conductive; poor biocompatibility with living systems)


XRD spectrum of electrochemically obtained graphene

Few-layer graphene- FLG

Multi-layer graphene- MLG

n= D/d- number of layers

Bragg’s law: nλ = 2d sinϴ

Scherrer equation: D = Kλ/βcosθ

Where: d = interlayer distance D = the mean size of the crystalline domains K is the shape factor, λ is the x-ray wavelength, β is the line broadening at half the maximum intensity (FWHM) in radians, and θ is the Bragg angle

5 10 15 20 25 30 35

0

10

20

30

40

50

Inte

nsit

y (

a.u

.)

2 (degrees)

Measured spectra

FLG

MLG

21

.28

25

.99

21.28 25.99

No. of layers 3 15

% 77 23 Gr 22a

5 10 15 20 25 30 35

0

10

20

30

40

Measured spectra

FLG

MLG

Inte

nsit

y (

a.u

.)

2 (degrees)

21.23 26.26

No. of layers 3 27

% 93 7

26

.26

21

.23

Gr 35

0.5 M- electrolyte

1 M- electrolyte

XRD spectra of electrochemically obtained graphene

Few-layer graphene- FLG

Multi-layer graphene- MLG



Elextrolytes: mixture of strong acids (sulfuric; nitric); salts (ammonium sulfate ) Advantages: - Prevent the over-heating of the solution - Less graphitic material in the final product

D) Exfoliation of graphite rods via pulses of current: sensitive detection of 8-hydroxy-2’-deoxyguanosine (8-OHdG)

EGr-A: mixture of sulfuric and nitric acid (3:1 ratio; 1 M each; 100 mL volume) EGr-S: 0.2 M ammonium sulfate

Experimental conditions Applied bias: 4-15 V Pulse duration: 0.8 s Pause: 0.2 s Time: 3 hours



Scherrer equation: D = Kλ/βcosθ

Where: d = interlayer distance D = the mean size of the crystalline domains K is the shape factor, λ is the x-ray wavelength, β is the line broadening at half the maximum intensity (FWHM) in radians, and θ is the Bragg angle

XRD spectra of the synthesized materials

Bragg’s law: nλ = 2d sinϴ

EGr-S EGr-A

n= D/d- number of layers

8-hydroxy-2’-deoxyguanosine (8-OHdG)

- 8-OHdG is an oxidative lesion of DNA induced by free radicals - its high concentration in body fluids is an indication of disease (cancer; periodontitis) - < 10-7 M – normal concentrations - 10-7 – 10-6 M – indication of a disease



CVs recorded with bare GC (black) and graphene modified electrodes, GC/EGr-A (blue), GC/EGr-S (red) in pH 6 PBS solution containing 10−4 M 8-OHdG; 10 mV·s−1 scan rate

0.0 0.2 0.4 0.6 0.8

-2.0x10-4

-1.0x10-4

0.0

1.0x10-4

2.0x10-4

3.0x10-4

4.0x10-4

0.53 V

0.48 V

GC

GC/EGr-A

GC/EGr-S

E(V) vs Ag/AgCl

i

(A/c

m2)

0.47 V

Detection of 8-OHdG with graphene-modified electrodes

EGr-S : graphene in ammonium sulfate EGr-A : graphene in acidic solution


0.0 0.2 0.4 0.6 0.81x10

-6

2x10-6

3x10-6

4x10-6

5x10-6

6x10-6

7x10-6

8x10-6

8-OHdG in PBS pH 6 PBS 6

10-7

M

3x10-7

M

6x10-7

M

10-6

M

3x10-6

M

6x10-6

M

10-5

M

3x10-5

M

6x10-5

M

10-4

M

I (A

)

E (V) vs Ag/AgCl

GC/EGr-A

0.47 V

a.

GC/EGr-S Linear range: 3 x 10-7 – 10-4 M Sensitivity: 0.67 A/cm2 M LOD: 9.8 x 10-8 M GC Linear range: 10-6 – 10-4 M Sensitivity: 0.22 A/cm2 M LOD: 3 x 10-7 M

Detection in standard laboratory solutions (pH 6 PBS)


Detection of 8-OHdG in artificial urine (pH 5.9)

GC/EGr-S Linear range: 3 x 10-7 – 10-4 M Sensitivity: 0.79 A/cm2 M LOD: 9.8 x 10-8 M GC Linear range: 10-6 – 10-4 M Sensitivity: 0.15 A/cm2 M LOD: 3 x 10-7 M


Reproducibility of GC/EGr-S electrodes

0.0 2.0x10-5

4.0x10-5

6.0x10-5

8.0x10-5

1.0x10-4

0.0

1.5x10-5

3.0x10-5

4.5x10-5

6.0x10-5

7.5x10-5

pH 6 PBS

C (M)

I peak (

A/c

m2)

GC/EGr-S1

GC/EGr-S2

GC/EGr-S3

a.

GC/EGr-S

0.0 2.0x10-5

4.0x10-5

6.0x10-5

8.0x10-5

1.0x10-4

0.0

2.0x10-5

4.0x10-5

6.0x10-5

8.0x10-5

b.

C (M)

I peak (

A/c

m2)

GC/EGr-S1

GC/EGr-S2

GC/EGr-S3

Artificial Urine

GC/EGr-S

Testing saliva samples with screen-printed electrodes

SALIVA • Water: 99.5% • Electrolytes

•2-21 mM sodium •10-36 mM potassium •1.2-2.8 mM calcium •0.08-0.5 mM magnesium •25 mM bicarbonate •1.4-39 mM phosphate

•Mucus •Antibacterial compounds (hydrogen peroxide; immunoglobulin A; thiocyanate) •Epidermal growth factor •Various enzymes

The presence of 8-OHdG in saliva indicate periodontitis (gum disease)

Screen-printed electrodes modified with graphene (EGr-S)

0.0 0.1 0.2 0.3 0.4 0.5 0.6-4.0x10

-6

-2.0x10-6

0.0

2.0x10-6

4.0x10-6

6.0x10-6

8.0x10-6

1.0x10-5

saliva

1x10-6

3x10-6

6x10-6

1x10-5

3x10-5

6x10-5

1x10-4

3x10-4

6x10-4

I (A

)

E (V) vs Ag/AgCl

0.34 V

DS SPEXF 30 III

0.0 2.0x10-4

4.0x10-4

6.0x10-4

0.0

1.0x10-6

2.0x10-6

3.0x10-6

4.0x10-6

5.0x10-6

6.0x10-6

Saliva

DS SPEXF 30

I pe

ak (

A)

C (M)

Electrolyte: saliva diluted five times with PBS pH 6 and spiked with 8-OHdG

Screen-printed electrodes Linear range: 3 x 10-6 – 10-4 M LOD: 10-6 M

50 microL

0.0 0.1 0.2 0.3 0.4 0.5 0.6

0.0

2.0x10-6

4.0x10-6

6.0x10-6

8.0x10-6

SPEXF21

I (A

)

E (V) vs Ag/AgCl

Cx

Adaos I

Adaos II

Adaos III

0.0 5.0x10-5

1.0x10-4

1.5x10-4

2.0x10-4

2.5x10-4

5.0x10-7

1.0x10-6

1.5x10-6

2.0x10-6

2.5x10-6

3.0x10-6

x = 6.449 x 10-7/0.00974 = 6.62 x 10

-5 M

I pe

ak

(A

)

C (M)

SP EXF 21

Xadded

= 6 x 10-5

M

Xfound

= 6.62 x 10-5

M

Conclusions Graphene can be obtained by various methods (CVD; chemical; electrochemical) The synthesis method clearly influences the electro-catalytic properties of graphene Biomarkers can be detected with such electrodes in laboratory solution More work is needed to detect the biomarkers in body fluids

Thank-you Dr. Alexandru R. Biris

Dr. Crina Socaci

Dr. Florina Pogacean

Dr. Maria Coros

Eng. Valentin Mirel

Dr. Eng. Stefan Gergely

Dr. Codruta Varodi

Dr. Marcela Rosu

Dr. Lidia Magerusan

Alex Turza, PhD student

Acknowledgment “Program Nucleu” MEN-Romania PN-09-44 01 15 and Project PN 18 03 02 02 CNCS/CCCDI-UEFISCDI, Romania Project PN-III-P2–2.1-PED-2016-0392 (102PED/2017) Project PN-III-P2–2.1-PED-2016-0415 (103PED/2017) Project PN-III-P4-ID-PCCF2016-0006 (PCCF20/2018)

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