Applications of Atmospheric Pressure

Non-thermal Plasmas in Medicine and Dentistry

Wei-Dong Zhu Saint Peter’s College

3rd Graduate Summer Institute “Complex Plasmas”

Seton Hall University, NJ, USA

August 8, 2012

My colleagues

Peng Sun, HongQing Feng, RuiXue Wang, Jie Pan, YiFeng Chai, Jue Zhang, Jin Fang

at Peking University, China Na Bai, Ye Tian, and FuXiang Liu at Sichuan University, China Jose Lopez at Seton Hall University, NJ, USA Kurt Becker at Polytechnic Institute of NYU, NY, USA Many more…

2

A joint effort 3

are well understood

are used extensively nowadays

(e.g. in semiconductor industry for

computer chips manufacturing)

Low pressure plasmas (1 mTorr ~ a few Torr)

However, to generate low pressure plasmas, it involves

vacuum chambers

expensive vacuum pumps

pressure monitoring and pressure control devices

Plasmas are easier to be generated

at low pressures

Generate Plasmas at Atmospheric Pressure!!

+ + =

4

What happens at air pressure?

• No vacuum is involved

• Difficult to generate and sustain

• Run into some challenges such as glow to

arc transition – Non controllable

Arc Discharge: thermal plasma

-It’s hot and detrimental

-Gas temperature can reach as high as 2x104 K

- Low voltage drop at cathode

- High cathode current density

5

Transient plasmas: In atmospheric plasmas, for efficient gas heating at least

100-1000 collisions are necessary. Thus, if the plasma duration is shorter than 10-

6 – 10-5 s, gas heating is limited. Of course, for practical purposes such plasma

has to be operated in a repetitive mode, e.g., in trains of microsecond pulses with

millisecond intervals.

Dielectric Barrier Discharges: These plasmas are typically created between

metal plates, which are covered by a thin layer of dielectric or highly resistive

material. The dielectric layer plays an important role in suppressing the current:

the cathode/anode layer is charged by incoming positive ions/electrons, which

reduces the electric field and hinders charge transport towards the electrode.

DBD also has a large surface-to-volume ratio, which promotes diffusion losses

and maintains a low gas temperature.

Micro-plasmas: Gas heating occurs in the plasma volume, and the energy is

carried away by thermal diffusion/convection to the outside. If the plasma has a

small volume and a relatively large surface, gas heating is limited.

How do we solve this problem? 6

Soup / Cocktail?

Atmospheric Pressure Plasma

Charged Particles

(Secondary) Reactive Species

Visible Light

Thermal Radiation

EM Field

UV / VUV Radiation

7

Multi-phase interaction

Applications of Atmospheric Pressure Non-Thermal Plasmas

Atmospheric

Pressure Non-

Thermal Plasmas

Atmospheric

Pressure Non-

Thermal Plasmas

8

Electrostatic Precipitation

Ozone Generation

Electromagnetic

Reflection, Absorption,

and Phase Shift

Enhancing

Hydrocarbon-Air

Combustion

Mitigation of the Shock Waves in

Supersonic/Hypersonic Flights

Surface Treatment

Chemical

Decontamination

Biological Decontamination

Plasma Medicine

Dentistry

Material

synthesis

Lighting Sensing

Plasma TV

…

Why Plasma Medicine?

Hospital-Acquired Infections (Year 2002 in US: >1.5 million cases, 99k deaths)

Poor hand hygiene compliance of health care workers

Catheter-associated infections (urinary tract and bloodstream)

Surgical site infections

Ventilatilator-associated pneumonia

…

Vs

Not a Fair Fight!

Microbes Humans Factor

# on earth 5x1031 7x109 ~1022

Mass (tons) 5x1016 3x108 ~108

Generation time 30 mins 30 years ~5x105

Time on earth (yrs) 3.5x109 4x106 ~103

Projan SJ., Curr Opin Microbiol 6 (2003) 427-430

9

Emerging Antimicrobial Resistance

Since 1940, antibiotics substantially reduced threat of infectious diseases

However, antimicrobial resistant microbes are emerging and spreading

10

Infection

First-line of antimicrobials (usually cheap)

Cured

No

Second-line drugs (more expensive)

Third-line drugs (much more expensive)

Yes

Yes

Yes No

No

Toxi

c To

xic

???

ISDA Is Concerned (Infectious Disease Society of America)

Spellberg et. al., CID, 38 (2004) 1279-1286

Pharmaceutical companies aren’t developing new drugs!

Atmospheric pressure, non-thermal plasmas are inexpensive, easy to generate

(in many forms and often as desired) – may be used against microbes

11

The Field of Plasma Medicine

(Inorganic) Surface

Treatment & Modification

Atmospheric Pressure Plasma

Sources

Blood Coagulation

Tooth Root Canal

Therapy

Air / Water Treatment

Wound Healing

Tooth Whitening

Cancer Therapy

Many More…

12

A Brief History of Plasma Medicine (Plasma Health Care)

1850s Siemens used a dielectric barrier discharge (DBD) to generate ozone and used the ozone to clean water from biological contaminants.

1960s to 1980s Some attempts were made to use plasmas for biological sterilization. Plasma was used as a secondary agent in the sterilization process, and no scientific investigations were made to understand how plasma actually interacted with bacterial cells and how it caused their demise.

1990s - Now Systematic research was conducted to understand the interaction between plasmas and biological cells. This includes effort in both atmospheric pressure and low pressure plasmas.

1997 First funding (AFOSR) for this kind of multidisciplinary research that bridged plasma physics and biology started.

2007 First International Conference on Plasma Medicine (every 18 months)

2009 International Society on Plasma Medicine established

Journal of Plasma Medicine launched

2012 Many research groups around the world are dedicating more and more of their effort in the research area of plasma medicine.

Funding opportunities around the world!!

13

An Incomplete list of Atmospheric Pressure Plasma Sources

14

You Are Unique, Just Like Everyone Else

Classification

Geometric appearance

Single jet plasma sources

Large area plasma sources

Direct vs. Indirect

Treated surfaces stand alone

Treated surfaces are part of the circuit

Hybrid

Electrical Power

DC, Pulsed DC

AC, RF, Microwave

Microhollow Cathode Discharge Plasma Micro Jet

15

Electrode

In contact with plasma

Not in contact (dielectric barrier)

DC MHCD Plasma Micro Jet 16

Plasma Micro Jet

[A. Mohamed, et al., US patent application 20060028145] [J. Kolb , et al., Applied Physics

Letters, 92, 241501 2008]

Metal

Insulator

5 kW

100 W - HVG

as F

low

Dimensions of the device are: Opening of inner electrode: Ø : 800 mm Opening of outer electrode: Ø : 400 -800 mm Separation: 500 mm Depth of exit opening: ~1 mm Electrode material: copper

He

He/O2

Air

17

DC MHCD Plasma Micro Jet

Air as the Working Gas

Inactivation of Bacteria on Petri Dish

Inactivation of Bacterial Spores on Petri Dish

Inactivation of Bacteria in Water

Inactivation of Bacterial Spores in Water

Teeth Whitening

18

Typical Working conditions

Working gas: Air

Flow rate: 2-3 slm

Sustaining voltage: 400-500 V

Discharge current: 20-30 mA

List of bacteria studied

Experimental Procedure Total path length: 120 mm Moving speed: 4 mm/s Time per path: 30 s Total treatment time: 30s / 60s / 90 s Area exposure/path: < 1 s (visible plasma), Culture preparation: standard procedure Inactivation analysis: counting of Colony Forming Units (CFUs)

Bacteria Gram

stain

A Escherichia coli Negative

B Staphylococcus

aureus Positive

C Micrococcus

luteus Positive

D Bacillus

megaterium Positive

E Bacillus subtilis Positive

F Bacillus natto Positive

Treated

Untreated

Inactivation of Bacteria on Petri Dish 19

E. coli

S. aureus

M. luteus

B. megaterium

B. subtilisB. natto

-5

0

5

10

15

20

25

30

35

40

Su

rviv

al

Rate

(%

)

Treated Area

Untreated Area

Survival Rate of Bacteria (Vegetative State)

Exposure Time: 90 s

0.0 0.5 1.0 1.5

0

20

40

60

80

100 E. coli

Untreated Area

Treated AreaSu

rviv

al R

ate

(%

)

Treatment Time (min)

0.0 0.5 1.0 1.5

0

20

40

60

80

100M. luteus

Treated Area

Untreated AreaSurv

ival R

ate

(%

)

Treatment time (min)

CFUSurvival Rate= 100%

CFU

survival

control

20

H. Feng et al., IEEE Transaction on Plasma Science, 37 (2009) 121

Plasma Dose Effect

E.coli

M. lu

teus

Control 30 seconds 60 seconds 90 seconds

radially decreasing survival rate uniform decreasing survival rate

21

H. Feng et al., IEEE Transaction on Plasma Science, 37 (2009) 121

Inactivation of B. subtilis Spore on Petri Dish

spores are more difficult to inactivate because of multiple

layers and coatings around the genetic core

B. subtilis spore0

10

20

30

40

50

60

Surv

ival R

ate

(%

)

Treated Area

Untreated area

Treating Time: 1.5 min

Survival rate of B. subtilis spores

(after 90 s totel treatment time)

0.0 0.5 1.0 1.5 2.0 2.5

0

20

40

60

80

100 B. subtilis Spores

Treated Area

Untreated Area

Surv

ival R

ate

(%

) Treatment Time (min)

B. subtilis spores survival rate for

various plasma treatment times

22

H. Feng et al., IEEE Transaction on Plasma Science, 37 (2009) 121

A few questions before moving on

• Are they really dead?

• How did the plasma do it?

– UV?

– Heat?

– Radicals?

– Combinative effect?

23

• UV – confined to treated area (because of rapid absorption)

• Reactive Radicals (O, OH, O3) – these species have a long life time and can

migrate into the untreated area (with radially decreasing concentration)

• Ions – measured ion current away from the nozzle suggests their presence in

and near the treated area

• Sequential/simultaneous action of UV, reactive radicals, and ions inactivates

the different bacteria/spores with varying efficacy

SEM of plasma treated (~1.5 min) S. aureus show clear poration on cell

membrane as well as the change of the cell morphology, which indicates

the modification of the cell after the plasma treatment.

Control 1.5 min PMJ treatment

Damage of Cell Membrane

SEM pictures of S. aureus before and after 1.5 min PMJ treatment

24

Plasma Evaluation

200 250 300 350 400 450 500 550 600 650 700 750 800 850

0

1000

2000

3000

4000

5000 UV

C

UV

A

Em

issi

on

In

ten

sity

(A

.U.)

Wavelength (nm)

UV

B

700 710 720 730 740 750 760 770 780 790 800

100

150

200

250

300

350

400

450

N2

N2

N2

Em

issio

n In

ten

sity (

A.U

.)

Wavelength (nm)

ON2

NO

N2

25

International Light

Calibrated Radiometer

calibrated detector

(covers 220–310 nm)

At a flow rate of 2 slm, current of 20 mA: ~300 μW/cm2

Temperature at 1 cm away from the exit nozzle: ~ 40 oC

Inactivation of Bacteria in Water

• Bacterium: S. aureus

• Liquid: 20 ml (contained in a 50 ml beaker)

(1) 20ml distilled water

(2) 19ml distilled water with 1ml LB culture media

(3) 19ml distilled water with 1ml bacteria suspension

• Working gas: compressed ambient air

• Flow rate: 2-3 SLM

• Power consumption: 8 W (400 VDC, 20 mA)

• Plasma Activated Species directly interact with Liquid Media

• Micro-liquid droplets in the gas bubbles from gas-liquid mixing increase the surface

area for the chemical reactions higher reaction efficiency

• No extra electrode or electrolysis in the liquid is needed

26

According to World Health Organization, 80% of

illnesses are caused by drinking polluted water, and

50% of the children deaths are also resulted from the

waterborne diseases.

Some of these pathogens are more resistant to

traditional methods of sterilization, such as heating,

chlorination and UV radiation

2min 4min 6min 8min 10min 0min

12min 14min 16min 18min 20min

27

Inactivation of S. aureus in Water

SEM pictures of S. aureus

before PMJ treatment in water after 20 min of PMJ treatment

Experiment were carried out with an initial pH value of 7.5

S. aureus underwent a transition from initially smooth surfaces to surfaces with a

single-dip after the PMJ treatment. This was not observed in the negative control

samples where only gas was introduced into the liquid without a plasma.

28

29

Inactivation of S. aureus in Water

-2 0 2 4 6 8 10 12 14 16 18 20 22

-10

0

10

20

30

40

50

60

70

80

90

100

110

120

Inactivation RateIn

activa

tio

n R

ate

(%

)

Time (min)

19ml distilled water with 1ml bacteria suspension

CFUInactivation Rate=(1- ) 100%

CFU

survival

control

F. Liu et al., Plasma Processes and Polymers, 7 (2010) 231

-2 0 2 4 6 8 10 12 14 16 18 20 22

12

16

20

24

28

32

36

40

44

48

Overall Water Temperature

20ml H2O

19ml H2O+1ml Sample

19ml H2O+1ml Culture

Tem

pera

ture

(oC

)

Time (min)

-2 0 2 4 6 8 10 12 14 16 18 20 22

2

3

4

5

6

7

8

Overall pH value of water

19ml H2O+1ml Sample

19ml H2O+1ml Culture

20ml H2O

pH

Valu

e

Time (min)

30

-2 0 2 4 6 8 10 12 14 16 18 20 22

-10

0

10

20

30

40

50

60

70

80

90

100

110

120

Inactivation Rate

pH

Valu

e

Inactiva

tio

n R

ate

(%

)

Time (min)

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

pH Value

O2

-• + H+ HOO•

pH=4.4

O2-• + H+ HOO•

pKa=4.8

Inactivation of S. aureus in Water F. Liu et al., Plasma Processes and Polymers, 7 (2010) 231

31

-2 0 2 4 6 8 10 12 14 16 18 20 22

-10

0

10

20

30

40

50

60

70

80

90

100

110

120

Inactivation Rate

pH

Valu

e

Inactivation R

ate

(%

)

Time (min)

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

pH Value

O2-• + H+ HOO•

pKa=4.8

Inactivation of S. aureus in Water (pH preset to 4.5)

-2 0 2 4 6 8 10 12 14 16 18 20 22

0

20

40

60

80

100

Inactivation Rate

pH Value

Time (min)

Inactivation R

ate

(%

)

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

pH

Valu

e

F. Liu et al., Plasma Processes and Polymers, 7 (2010) 231

No charges - Easier to

penetrate cell (check

Hamaguchi’s)

Bacillus Subtilis Spores

• B. subtilis, can form a tough protective endospore, with several protein-based outer layers and cortex peptidoglycan and a compact genetic core, allowing the organism to tolerate extreme conditions such as high temperature, strong acidic/alkaline environments.

• It is reported that the necessary UV dosage to inactivate B. subtilis spores is 9 times of that is required for E. coli.

• The intrinsic resistance and special spore coat make it one of the most resistant bacteria to biocides.

• It is estimated that B. subtilis spores are 400 times more resistant to chlorine than enteric bacteria (rod-shaped Gram-negative bacteria) do.

• While overdose can in fact kill the spores, long term usage of biocide (including antiseptics, disinfectants and preservatives) can lead to bacteria biocide resistance.

32

33

Inactivation of B. subtilis Spores in Water

0 2 4 6 8 10 12 14 16 18 20 22 24

0

20

40

60

80

100

Inactivation Rate

pH Value

Water Temperature

Time (min)

Ina

cti

va

tio

n R

ate

(%

)

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

pH

Va

lue

25

30

35

40

45

50

Te

mp

era

ture

(oC

)

100 nm

P. Sun et al., Plasma Processes and Polymers 9 (2012) 157 – 164

34

Inactivation of B. subtilis Spores in Water

100 nm

-2 0 2 4 6 8 10 12 14 16 18 20 22

0

20

40

60

80

100

120

Ina

cti

va

tio

n R

ate

(%)

Time (min)

pH=3

pH=2

pH=1

P. Sun et al., Plasma Processes and Polymers 9 (2012) 157 – 164

pH decrease in the plasma-water system is

not the main reason for the inactivation

Evaluation of Reactive Species

Evaluation of the reactive species in the liquid (long or short lived)

• H2O2 drop count titration kit (thiosulphate method)

[H2O2] (the kit may also react with other long lived oxidative species (e.g. O3))

• High Performance Liquid Chromatography (HPLC)

[NO3-]/[NO2

-], [Cu+]/[Cu2+]

• Optical Emission Spectroscopy (OES)

Identify gas phase excited species (e.g. O, OH, N, N2 and N2+)

• Electron Spin Resonance (ESR) Spin-trap Spectroscopy

0

10

20

30

40

42.16

20 min

NO

- 3 C

on

ce

ntr

atio

n (

mg

/L)

0 min

0.7

35

200 250 300 350 400 450 500 550 600 650 700 750 800 850

0

1000

2000

3000

4000

5000

6000

700 710 720 730 740 750 760 770 780 790 800

100

150

200

250

300

350

400

450

N2

N2

N2

Em

issi

on Inte

nsi

ty (A

.U.)

Wavelength (nm)

ON2

NO

N2

N+ 2(B

2

+ u-X

2

+ g)

N2(C

3

B

3

g)

Em

issio

n In

ten

sity (

A.U

.)

Wavelength (nm)

NO-

Electron Spin Resonance Spectroscopy

ESR no magnet with magnet

microwave

unpaired

electron

paired

electron

B

Radical in liquid

(short lifetime)

+

Spin trapping chemical

||

Spin adduct (longer lifetime)

Spin Trapping

Short-lifetime radicals in liquid can be identified indirectly Courtesy of S. Hamaguchi

36

e BE g Bm

h E

Spin-Trapping ESR: Singlet Oxygen

3325 3350 3375 3400 3425 3450 3475 3500

-400

-200

0

200

400

-400

-200

0

200

400

Magnetic Field(Gauss)

20 mg L-His(b)

Sig

nal In

ten

sitym

(A.U

.)

TEMPOaN

(a)

37

• Singlet molecular oxygen (in particular, O2

(1Δg)) has high oxidization ability and attacks

the double bonds of unsaturated fatty acid on

cell membrane, initiating lipid peroxidation

• Relatively longer half-life (~ 2 μs) in water.

• 2,2,6,6- tetramethylpiperidine (TEMP) was

used as spin trap of singlet oxygen, resulting in

spin adduct TEMPO (triplet pattern in ESR

spectrum with peak ratio 1:1:1 and hyperfine

splitting constant aN=1.736 mT).

• L-His (a O2 (1Δg) quencher) into the system

prior to the plasma treatment. TEMPO signal

decays with the increase of L-His and

completely diminishes when 20 mg L-His is

added into the system

Spin-Trapping ESR: Hydroxyl Radical

3350 3400 3450 3500-800

0

800

-800

0

800

-800

0

800

Magnetic Field(Gauss)

100 U SOD

0.225 mmol Mannitol

(c)

(b)

Sig

nal In

tensity (

A.U

.)

DMPO-OH(a)

38

• Hydroxyl radical (•OH) - oxidation potential of

2.8 V, is most reactive and toxic species

among all ROS.

• Short lifetime in water: ~ ns

• 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)

was used to spin trap •OH, resulting in spin

adduct DMPO-OH (a quartet pattern in ESR

spectrum with peak ratio 1:2:2:1, and

superfine splitting constants aN=aH=1.49 mT).

• When 0.255 mmol mannitol (a specific

scavenger of •OH), was added into the

solution before plasma treatment, DMPO-OH

signal was attenuated, indicating that the

majority DMPO-OH adducts derived from the

direct combination of DMPO and •OH.

DMPO DMPO-OH

Spin-Trapping ESR: Superoxide Anion 39

• DMPO can in principal spin trap superoxide

anion (•O2-), too. The spin-trap adduct

DMPO-OOH (the combination of DMPO with

HOO•, the conjugate acid of •O2-) usually

shows a 12-peak pattern in ESR spectrum

None was observed!

Four possible reasons:

The reaction rate constant between DMPO

and •OH (109 M-1 sec-1) is much larger than

that between DMPO and •O2- (lower than 104

M-1 sec-1 at pH 5);

The half-life of DMPO-OH (870 seconds) is

much longer than that of DMPO-OOH

(around 60 seconds)

DMPO-OOH tends to quickly decay to

DMPO-OH.

In water, •O2- be coverts to •OH via a few

reactions:

2 2 22 (self dismutation)O e H H O 2

2 2 (Fenton reaction)H O Cu OH OH Cu 2

2 2 (Oxidation and reduction)Cu O Cu O

2 2 2 2 (Haber-Weiss reaction)O H O OH OH O

3350 3400 3450 3500-800

0

800

-800

0

800

-800

0

800

Magnetic Field(Gauss)

100 U SOD

0.225 mmol Mannitol

(c)

(b)

Sig

nal In

tensity (

A.U

.)

DMPO-OH(a)

Evaluation of H2O2

• H2O2 in deionized water treated by a PMJ was evaluated with a hydrogen peroxide test kit (Model HYP-1; HACH Company, Loveland, Colorado, USA): ~ 80 mg/L after 15 minutes of plasma treatment.

• In premixed H2O2 solutions, only when the H2O2 concentration in water reached 800 mg/L, did we observe an average inactivation rate of 80% for S. aureus.

• H2O2 produced by the PMJ in water plays a lesser important role in the inactivation process of bacteria in the current system.

40

0 200 400 600 800

0

20

40

60

80

100

Inactiva

tion

Rate

(%

)

H2O

2 Concentration (mg/L)

Aesthetics of the tooth, especially tooth color is of

great importance to people.

Eating habit, smoking, drugs and chemicals can cause unfavorable tooth stain

Smile Factory: Healthy smile promotes social

communication

Dental fluorosis Tetracyline teeth External stain

41

Teeth Whitening (Bleaching)

Organic compounds that possess extended conjugated chains of

alternating single or double bonds and often include heteroatoms,

carbonyl, and phenyl rings in the conjugated system and are often

referred to as a chromophore.

Andrew Joiner, Journal of Dentistry, 34(2006)412–419

J.E. Dahl，U. Pallesen，Crit Rev Oral Biol Med，14(4):292-304 (2003)

Contemporary tooth whitening system are based primarily on

hydrogen peroxide

H2O22HO.

HO.+ H2O2 H2O+ HO2.

HO2. H+ + O2

.-

2H2O2 2H2O + 2{O} 2H2O + O2

H2O2H+ + HOO-

42

Teeth Whitening

• Working Gas: Air

• Flow Rate: 1.5 slm

• Distance between exit nozzle and Tooth surface: 1 cm

• Teeth Whitening Gel (beyondTM)

Active ingredient: H2O2 (35%)

• Gel replenished every 30 seconds

“No thermal-damages”

43

Prolonged exposure of teeth to temperature higher than 42 °C (critical temperature) may cause thermal injury to bone

0 2 4 6 8 10 12 14 16 18 20 22

16

18

20

22

24

26

28

30

32

34

36

38

40

To

oth

Su

rface T

em

pera

ture

(oC

)

Time(min)

Plasma Assisted Teeth Whitening

before after 20min

H2O2 before after 20min

Plasma+

H2O2 (35%)

Whitening Results

L*: Lightness

a*: Redness-Greenness

b*: Yellowness-Blueness

CIE(L*,a*,b*) color

classification scheme

0.0

2.0

2.5

3.0

3.5

4.0

H2O

2, 38

oCH

2O

2, PMJ

E

H2O

2, RT

44

Control Plasma + H2O2

The oxidation-reduction of Hydrogen Peroxide could influence the tooth surface structure when treat the teeth surface 20min.

Cold plasma could enhance the oxidation-reduction of Hydrogen Peroxide

H2O2

250×

1000×

Acid pickling Roughness

Surface Morphology 45

Clinical Treatment

(clinical gel + light )

It gets even better – No need for H2O2 at all 46

Saline + air blow

Saline + plasma

H2O2 (35%) only

1

2

3

4

5

6

H

2O

2 gel

Group C

Saline+PMJ

Group B

Saline+air blow

Group A

* av

g

J. Pan et. al., IEEE Transaction on Plasma Science, 38 (2010) 3143

Summary I

Atmospheric pressure non-thermal air plasma micro jet can

– effectively inactivate bacteria (gram positive and gram negative) on Petri dish both in the treated zone and untreated zone

– effectively inactivate bacterial spores on Petri dish in the directly treated zone, not so much in the untreated zone

– effectively inactivate bacteria and bacterial spores in water

– enhance tooth whitening effect of dental gel with acceptable morphological modification of enamel surface

He/O2 (2%) as Working Gas

Inactivation of Planktonic Fungi on Microtiter Plate

Inactivation of fungal biofilm

Eukaryotic Response Mechanisms

Anti-oxidative Defense of Cells

Inactivation of S. aureus in Water

Inactivation of Planktonic Fungi in Water

Study of Reactive Oxygen Species in Water

48

Why He/O2?

300 400 500 600 700 800

0

50

100

150

200

250

300

350

304 308 312 650 660

He

50

1

He

58

7

He

66

7

He

72

8

O 8

44

Em

issio

n Inte

nsity (

A.U

.)

Wavelength

O 7

77

He

70

6

He

49

2H

e 4

71

He

+ 4

68

Cu

+ 3

89

Cu

32

5C

u 3

27

OH (A-X)

x250

H

65

6x1

00

A typical optical emission spectrum of the

plasma jet with He/O2 (2%) as working gas

Strong atomic oxygen emission

*

2 2He O He O e

Penning Ionization (Ionization energy ~12 eV)

Electron impact dissociation

2 2O e O

Lower UV irradiance

comparing to air as working

gas (at the same current

and gas flow rate): ~ 1/10

49

Easier to ignite (lower voltage)

More stable operation

Inactivation of Staphylococcus aureus (S. aureus) in water

0 2 4 6 8 10

0

20

40

60

80

100

Ina

ctiva

tio

n R

ate

(%

)

Time (min) 0 min 4 min

8 min 16 min

50

Starting Concentration: 104 CFU/ml

Liquid: distilled water (20 ml)

Sustaining Voltage: 400 V

Discharge Current: 35 mA

LIVE/DEAD® BacLight™

Bacterial Viability Test

N. Bai et. al., Plasma Processes and Polymers, 8 (2011) 424

Reactive Oxygen Species

0 2 4 6 8 10

2

3

4

5

6

7

8

Time (minutes)

pH

Va

lue

pH

20

22

24

26

28

30

32

34

Te

mp

era

ture

(oC

) Temperature

3360 3400 3440 3480

-1500

-1000

-500

0

500

1000

1500

Magnetic Field (Gauss)

T

EM

PO

In

ten

sit

y (

A.U

.) (a)

0.00 0.03 0.06 0.09 0.12

0

300

600

900

1200

1500

L-His (mmol)

TE

MP

O In

ten

sit

y (

A.U

.) (b)

Singlet Oxygen

3360 3400 3440 3480-1500

-1000

-500

0

500

1000

Magnetic Field (Gauss)

DM

PO

-OH

In

ten

sit

y (

A.U

.) (a)

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

0

300

600

900

1200

1500

DM

PO

-OH

In

ten

sit

y (

A.U

.)

D-Man (mmol)

(b)

Hydroxyl Radical

It’s not the temperature!

It’s not the pH!

UV is doubtful!

H. Wu et. al., Plasma Processes and Polymers, 9 (2012) 417 - 424

51

0 20 40 60 80

0

200

400

600

800

DM

PO

-OH

In

ten

sit

y (

A.U

.)

SOD (U)

Which one is more important?

H. Wu et. al., Plasma Processes and Polymers, 9 (2012) 417 - 424

52

0

10

20

30

40

50

60

PMJ+L-His

(0.15 mmol)

PMJ+D-Man

(0.15 mmol)

PMJ+SOD

(200 U)

Su

rviv

al R

ate

(%

)

PMJ+H2O

CFUSurvival Rate= 100%

CFU

survival

control

Starting concentration: ~104 CFU/ml

Volume of liquid: 1 ml

Treatment time: 20 seconds

The addition of 200 U SOD has little

influence on the survival rate, although with

this amount of SOD, DMPO-OH signal

completely disappears. Neither ·O2- nor

·OH plays important roles.

D-Man is excellent in quenching ·OH

(2.7×109 M-1·sec-1) and weak in quenching 1O2 (<103 M-1·sec-1). When 0.15mmol D-

Man was added, DMPO-OH signal was

completely quenched, the survival rate rises

back to 13.4%, further verifies that ·OH

contributes little to sterilization.

L-His scavenges both ·OH (5×109 M-1·sec-

1) and 1O2 (3.2×107 M-1·sec-1). With the

addition of 0.15mmol L-His, TEMPO signal

was completely quenched, the survival rate

of S. aureus reaches ~40%

1O2 is more important than ∙OH and ∙O2

- in

the inactivation of S. aureus.

Antifungal Therapy

• Candidiasis, casued by Candida species, is the most common fungal infection in humans.

• Invasive diseases such as candidemia and candidiasis in deep-seated organ, mucocutaneous disorders such as oral candidiasis, vaginal and vulvovaginal candidiasis

• Have become a major clinical problem.

• Candida species exhibits planktonic unicellular form, but filamentary growth or complex multicellular structure is observed mainly in the infected tissues

53

Therapeutic strategies against Candida infection are very limited nowadays - systematic use of azole or topical use of imidazole.

Frequent prophylactic use of these drugs increases the infections caused by the drug-resistant Candida spp. strains such as C. krusei, C. glabrata, and fluconazole-resistant C. albicans strains, which can usually result in the treatment failure of commonly used antifungal agents such as fluconazole or other azoles

- HVG

as F

low

In Air

1 cm • DC “high voltage”: 400-500 V

• Current: 30-35 mA

• Working gas: helium/oxygen (2%)

• Starting concentration: 104 CFU/ml

• Sabouraud dextrose agar (SDA)

• Treated area: 2 cm x 2 cm

• Treatment time: 0 – 10 minutes

•

Strain

C. albicans (BMU 02971)

Resistant to fluconazole C. krusei (BMU 00279)

C. glabrata (BMU 00271)

C. albicans (SC 5314) Susceptible to fluconazole

Each path takes about 30 s Inactivation Rate = (1- ) 100%treated

control

CFU

CFU

Inactivation of Candida Strains on Petri Dish

40 oC

54

Inactivation of Candida Strains on Petri Dish

0 2 4 6 8 10

0

20

40

60

80

100

120

Ina

ctiva

tio

n r

ate

(%

)

Time (min)

C. glabrata 00271

C. krusei 00279

C. albicans 002971

C. albicans SC 5314

(a)

In Air (Treated Area)

• Treated area

o Fast increase of the inactivation rates within the first minute

o Different strains respond to plasma treatment differently

0 2 4 6 8 10

-20

0

20

40

60

80

100

Ina

ctiva

tio

n r

ate

(%

)

Time (min)

C. glabrata 00271

C. krusei 00279

C. albicans 002971

C. albicans SC5314

(b)

In Air (Untreated Area)

• Untreated area

o Initial change of the inactivation rate is not as prominent

o significant inactivation rate (~90%) achieved after a 10 minute

plasma treatment

P. Sun et. al., Applied Physics Letters, 98 (2011) 021501

55

r

R+/- HV

Ga

s F

low

Gas Cavity

Gas / Liquid

Interface

Micro liquid droplet

Gas

Liquid

In water

Inactivation of Candida Strains in Water

• Starting concentration: (1-3)×106 CFU/ml.

• 20 ml suspension was treated with He/O2 PMJ

• Treatment time: 0 to 4 minutes

• 200 μl suspensions was aspirated out and further

diluted 1000 and 100 times to perform XTT assay

and colony counting, respectively

0 1 2 3 4

0

20

40

60

80

100

120

Inac

tivati

on

rate

(%

)

Time (min)

C. glabrata 00271

C.kruseil 00279

C. albican 002971

C. albican SC5314

In Water

0 1 2 3 4

-20

0

20

40

60

80

100

120

XT

T(%

)

Time(min)

C. glabrata 00271

C. krusei 00279

C. albicans 002971

C. albicans SC5314

XTT assay

Antifungal susceptibility test

and cell viability test (2,3-bis(2-methoxy-4-nitro-

5-Sulfophenyl)-5-[(phenylamino)carbonyl])

P. Sun et. al., Applied Physics Letters, 98 (2011) 021501

56

Biofilm

http://www2.erc.montana.edu

57

What Are Biofilms

• Biofilms are complex microbial communities attached to a surface and embedded in a matrix composed mostly of extracellular

polysaccharides. They are ubiquitous!

• A biofilm community can be a single bacterial species, or in nature biofilms almost always consists of rich mixtures of many species of bacteria, as well as fungi, algae, yeasts, protozoa, other microorganisms, debris and corrosion products.

All Images taken from Center for Biofilm Engineering

http://www2.erc.montana.edu

From a reverse osmosis membrane

Teeth plague In a stream

In pipe section

58

How Are Bio-films Formed

1. Initial attachment of planktonic cell to the surface

2. Cell Proliferation

3. Accumulation in multilayer cell clusters

4. Maturation of biofilm architecture

5. Dispersion of bacterial cells from the biofilm

Iñigo Lasa, INT. MICROBIOL. v.9 n.1 Madrid mar. 2006

59

Inactivation of Candida Biofilms

Isolates Source Species BMU00279 Sputum (ATCC6258)

C. krusei BMU05102 Oral mucosa

BMU05137 Oral mucosa

BMU00271 Blood

C. glabrata BMU01689 Knee

BMU04388 Intraperitoneal fluid

BMU02971 Pharynx

C. albicans BMU03213 Oral mucosa

BMU04801 Oral mucosa

SC5314 Blood (Sequencing Strain)

• Strains grow on potato dextrose agar (PDA) at 35 ℃ for 3

days

• a loopful of Candida cells was inoculated in 20 ml yeast

peptone dextrose liquid medium and incubated overnight in a

shaker (150 rpm) at 30 ℃.

• Cells were then harvested from the liquid cultures by

centrifugation (3000 g×5 min at 4 ℃), and washed by ice-cold

sterile PBS.

• RPMI-1640 medium was used to re-suspend the pellet and

adjust the final density to 1.0×106 cells/ml for all strains.

• 100 ml of prepared suspension was pipetted into selected wells

of 96-well microtiter plates and every replication was

separated by an empty well.

• Biofilm was formed after incubation at 37 ℃ for 24 hours.

The medium was aspirated in the wells which were then

washed with sterile water three times to remove planktonic

cells.

• The biofilms were treated by PMJ for 10 seconds, 20 seconds,

30 seconds and 1 min, respectively.

Y. Sun et. al., Plos One, 7 (2012) e40629

60

Inactivation of Candida Biofilms

0 10 20 30 40 50 60

0

20

40

60

80

100

0

20

40

60

80

100

Time (s)

XT

T (%

)

BMU 05102

BMU 00279

BMU 05137

C. krusei

(a)

Ina

cti

va

tio

n R

ate

(%)

0 10 20 30 40 50 60

0

20

40

60

80

100

0

20

40

60

80

100

XT

T (%

)

Time (s)

BMU 01689

BMU 04388

C. glabrata

BMU 00271

(b)

Ina

cti

va

tio

n R

ate

(%)

0 10 20 30 40 50 60

0

20

40

60

80

100

0

20

40

60

80

100

Time (s)

BMU 02971

XT

T (%

)

C. albicans

SC 5314

(c)

BMU 04801

Inac

tivati

on

Rate

(%)

BMU 03213

Three important observations:

(1) different Candida biofilms (even the same strain isolated from different

sources) respond to plasma treatment slightly differently;

(2) all Candida biofilms are inactivated by PMJ rather quickly, all reaching

approximately 100% within 30 seconds.

(3) XTT assay corresponds well with CFU counts, indicating the loss of viability

of the cells.

Y. Sun et. al., Plos One, 7 (2012) e40629

61

SEM Images of Biofilms (C. albicans SC5314 as an example

Gas flow only 30 s 20 s 10 s

60 s

• With the increase of the plasma treatment time, cell

rupture, distortion and shrinking were observed.

• The biofilm lost its original morphological characteristics

and degraded to clusters of cell fragments.

• This degradation leads to the leakage of cell inclusion,

and therefore is detrimental for the survival of the fungi.

Y. Sun et. al., Plos One, 7 (2012) e40629

62

Antifungal Susceptibility Test

Two major concerns that motivated this test are:

(1) Some fungi may survive a certain dosage of plasma treatment. They are

possibly, however, modified by the plasma and are more susceptible to

traditional antifungal treatment.

(2) Clinical trials often combine traditional treatment methods and the

developing technique. As the toxicity and safety study of the plasma

treatment is still lacking, one might want to reduce the exposure to plasma to

a minimum dosage while achieving a considerable reduction of the dosage

of traditional antifungal therapy.

63

Antifungal Susceptibility Test

0 10 20 30 0 10 20 30 0 10 20 30

0

50

100

150

200

250

BMU 04801

C. krusei

BMU 05137

(c)(b)

C. glabrata

BMU 00271

(a)

C. albicans

SC 5314 BMU 00279 BMU 04388

BMU 02971 BMU 05102

BMU 01689

BMU 03213

Flu

co

na

zo

le (μ

g/m

l)

Times(s)

0 10 20 30 0 10 20 30 0 10 20 30

0

1

2

3

4C. albicans

BMU 04801

Time (s)

C. krusei

BMU 05137

C. glabrata

BMU 00271

(f)(e)

SC 5314

(d)

BMU 05102

BMU 04388

BMU 02971

BMU 00279

BMU 01689

BMU 03213

Am

ph

ote

ric

in B

(μ

g/m

l)0 10 20 30 0 10 20 30 0 10 20 30

0.0

0.4

0.8

1.2

1.6

2.0

(i)(h)

C. albicans

BMU 04801

(g)

C. krusei

BMU 05137

Time (s)

C. glabrata

BMU 00271

SC 5314 BMU 00279 BMU 04388

BMU 02971 BMU 05102

Ca

sp

ofu

ng

in (

μg

/ml) BMU 01689

BMU 03213

• Antifungal susceptibility test was performed in samples treated with PMJ following

Clinical and Laboratory Standards Institute recommended reference standard M27-A3

with minor modification.

• The sessile minimum inhibitory concentration (SMIC50) is defined as the antifungal

concentration at which a 50% decrease in absorbance was detected in comparison

with the control sample.

• Significant reductions of SMIC50 are observed for all plasma treated Candida biofilms.

• SMIC50 ranges from 1-16 μg/ml for fluconazole, 0.125-0.25 μg/ml for amphotericin B

and 0.015-0.5 μg/ml for caspofungin after a 10 s plasma treatment. These values are

reduced to ≤1μg/ml, ≤0.125μg/ml and ≤0.015μg/ml, respectively, after 20 and 30 s

treatments. Y. Sun et. al., Plos One, 7 (2012) e40629

64

H. Feng, Applied Physics Letters, 97 (2010) 131501

Eukaryotic Response Mechamism

Saccharomyces cerevisiae single gene mutants

65

Genes from both pathways are needed for the eukaryotic cells to

survive the plasma treatment

Summary II

• Atmospheric pressure non-thermal He/O2 (2%) plasma micro jet can

– effectively inactivate bacteria and fungi on Petri dish and in water

– effectively inactivate fungal biofilms

• Similar ROS but no pH effect

• Eukaryotic cells respond to plasmas at genetic level

66

Ar/O2/N2 as Working Gas

Assessment of the Roles of

Various Inactivation Agents

Tooth Root Canal, Dentinal Tubules

Infection and Re-infection Prevention

67

Inactivation of Staphylococcus aureus in water 68

pure argon,

argon with 2% oxygen,

argon with 2% oxygen and 10% nitrogen

(All research grade)

0 2 4 6 8 1010

2

103

104

105

106

To

tal C

FU

in

20

ml s

ys

tem

Time (min)

Ar/O2

Ar/O2/N

2

Ar

Control Ar

Ar/N2/O2 Ar/O2

10 min treatment

Q. Zhang et. al., Journal of Applied Physics, 111 (2012) 123305

Optical Emission Spectra

Q. Zhang et. al., Journal of Applied Physics, 111 (2012) 123305

69

Further evaluations

DMPO-OH

TEMPO

(DETC)2-Fe2+-NO

Q. Zhang et. al., Journal of Applied Physics, 111 (2012) 123305

70

Comparison of Different Species

Operating Gas [Ar/O2/N2] [Ar/O2] [Ar]

Inactivation Efficacy 2 1 3

Reactive Species

O 2 1 3

NO 1 - -

OH(OES) - - 1

OH(ESR) 1 2 3 1O2 1 2 3

H2O2 1 2 3

O3 (detected in air) 1 2 3

Cu+/Cu2+ 1 2 3

Others

Temperature 1 2 3

Acidity change 1 2 3

Voltage 1 2 3

71

Q. Zhang et. al., Journal of Applied Physics, 111 (2012) 123305

Inactivation of E. faecalis in human tooth root canal

Group Treatment Method Plasma

on/off Treatment Time

Re-Infection

Evaluation

Part I A Ar/O2 gas flow Off 0-8 minutes (2 min increment) No

B Ar/O2 PMJ On 0-8 minutes (2 min increment) No

Part II

C Ar/O2 gas flow Off 0 minute Yes

D Formocresol (FC) Off 7 days Yes

E Camphor Phenol (CP) Off 7 days Yes

F Ca(OH)2 Off 7 days Yes

G Ar/O2 PMJ On 10-40 minutes (10 min increment) Yes

72

0

20

40

60

80

100

120

2 4 6 8

In

ac

tivati

on

Ra

te(%

)

Time (mins)

Single-rooted extracted intact permanent teeth.

The root canals were prepared with Ni–Ti hand files

(Mani. Inc., Japan) up to size #40 following step-back

technique, and irrigated every time when file size was

changed to remove debris.

Each apical foramen was sealed with composite resin

for the inoculation of bacteria inside the root canal.

All specimens were sterilized by an autoclave before

further treatment. 10 μL fresh diluted E. faecalis

suspension (106 CFU/mL) was injected into root canals.

R. Wang, et. al., Plasma Medicine, 1 (2011) 143 - 155

Middle Third Ro

ot

Can

al

Apical Third

Group root canal (×100) Middle third (×5000) Apical third (×5000)

A

B

G

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

SEM images 73

R. Wang, et. al., Plasma

Medicine, 1 (2011) 143 - 155

Reinfection Evaluation

Samples were treated with different intracanal

medicament (formocresol (FC), camphor

phenol (CP) and Ca(OH)2) for 7 days

Samples were treated with PMJ for different

times (8, 10, 20, 30, 40 minutes).

All samples were temporarily sealed with

Cavit (3M ESPE, USA) and incubated at 37 oC

for one week.

The root canals were then rinsed and incubated

following the same procedure for re-infection

evaluation 0

20

40

60

80

100

8 min

Control

40min

PMJ

FCCa(OH)2CP30min

20min

10min

Re-i

nfe

cti

on

rate

(%)

treated controlRe-infection%=CFU /CFU 100%

o Root canal treated with PMJ for 8 minutes showed re-infection rate about 62% after 7 days.

o The re-infection rate decreased gradually with the increase of the PMJ treatment time,

reaching 0.8% for that with 30-min PMJ treatment. The samples treated with PMJ for 40

minutes presented no any re-infection.

o Samples treated with intracanal medicaments for 7 consecutive days showed re-infection

rate of 0% (FC), 2.8% (CP) and 4.2% (Ca(OH)2), respectively.

74

R. Wang, et. al., Plasma Medicine, 1 (2011) 143 - 155

Summary III

Atmospheric Pressure non-thermal Ar/O2/(N2) plasma micro jet can

– disinfect tooth root canal in 40 minutes with no re-infection

– Effectively inactivate E. Faecalis biofilms

Different chemistry: atomic oxygen plays an important role

75

Concluding Remarks

The field of plasma medicine and dentistry (or whatever names

people are sticking to) is young and promising

Standards need to be made to compare different inactivation

agents (challenging but inevitably has to be done)

Fundamental studies (including toxicity evaluation) are

extremely important

New tools for charactering plasma at the complex plasma-

liquid-solid surface need to be developed

76

We have to be careful of where we are going, otherwise, we may not get there!

Acknowledgement

China International Science and Technology

Cooperation under Grant number 2008KR1330

Electro-Energetic Physics Program of the U.S. Air

Force Office of Scientific Research (AFOSR) under

grant number FA9550-08-1-0332

Bioelectrics Inc.

Peking University Biomed-X Foundation

77

American Chemical Society – Petroleum Research Fund