Air plasma effect on dental disinfection

PHYSICS OF PLASMAS 18, 073503 (2011)

Air plasma effect on dental disinfection S. Duarte,1 S. P. Kuo,2 R. M. Murata,1 C. Y. Chen,2 D. Saxena,1 K. J. Huang,2 and S. Popovic3

1 Department of Basic Sciences and Craniofacial Biology, College of Dentistry, New York University, New York, New York 10010, USA 2 Department of Electrical and Computer Engineering, Polytechnic Institute of New York University, Brooklyn, New York 11202, USA 3 Department of Physics, Old Dominion University, Norfolk, Virginia 23529, USA

(Received 11 April 2011; accepted 8 June 2011; published online 21 July 2011) A nonthermal low temperature air plasma jet is characterized and applied to study the plasma effects on oral pathogens and biofilms. Experiments were performed on samples of six defined microorganisms’ cultures, including those of gram-positive bacteria and fungi, and on a cultivating biofilm sample of Streptococcus mutans UA159. The results show that the plasma jet creates a zone of microbial growth inhibition in each treated sample; the zone increases with the plasma treatment time and expands beyond the entire region directly exposed to the plasma jet. With 30s plasma treatment twice daily during 5 days of biofilm cultivation, its formation was inhibited. The viability of S. mutans cells in the treated biofilms dropped to below the measurable level and the killed bacterial cells concentrated to local regions as manifested by the fluorescence microscopy via the environmental scanning electron microscope. The emission spectroscopy of the jet indicates that its plasma effluent carries an abundance of reactive atomic oxygen, providing catalyst for the C 2011 American Institute of Physics. [doi:10.1063/1.3606486] observed plasma effect. V

I. INTRODUCTION

Plasma is an ionized gas composed of electrons, ions, and neutral particles; electrons can be energized by the electric field to produce free radicals, which have unpaired electrons on an open shell configuration. With some exceptions, the unpaired electrons cause radicals to be highly chemically reactive and capable of destroying a broad spectrum of microorganisms, in particular, by those oxygen associated radicals. Considerable research efforts have been devoted to applying plasmas for medical applications in recent years.1–9 Accordingly, there are considerable interests in developing plasma sources, which generate nonthermal plasmas, or “cold plasmas” at or near atmospheric pressures. The free radicals involving predominantly singlet, metastable molecular oxygen and nitrogen species, and reactive atomic oxygen (RAO), which is electronically excited atomic oxygen in more reactive form, produced in the plasma effluents can work effectively for prevention or treatment of infection diseases. Basically, there are three types of low temperature plasma jet or torch devices, categorized by the feeding gases that can generate oxygen and nitrogen radicals directly or indirectly. The first one uses pure rare (noble) gas, such as Argon10 or Helium,11 as the feeding gas; the noble gas requires lower discharge voltage so that the produced plasma can be kept at lower temperature. The oxygen and nitrogen radicals are generated in the ambient air by the plasma jet. The second, one is fed with a mixture of air (or oxygen) and helium gas;12,13 the energetic electrons from the electric discharge in the helium gas excite oxygen and ambient air into radicals. The third, one is fed with air or nitrogen only;4–9,14,15 it produces an air or a nitrogen plasma torch or jet, which can produce the most abundant radicals in the plasma efflu1070-664X/2011/18(7)/073503/7/$30.00

ent. For oral applications, there will be another constraint on the physical size of the plasma generator. Dental caries is among the more prevalent chronic human infections disease. The World Health Organization’s reports describe that caries prevalence is at 60%–90% in school-age children and virtually universal among adults.16 The etiology and pathogenesis of dental caries are generally ascribed to the colonization of tooth surfaces by multispecies biofilm containing mutans streptococci.17 A biofilm is a dense aggregation of microbial cells bound together by an extracellular matrix of polysaccharide and protein. When bacterial groups aggregate in these multicellular communities, they are able to tolerate antimicrobial challenges that normally eradicate free-floating individual cells.18 Polysaccharides promote adherence and accumulation of cariogenic streptococci on the tooth surface, enhancing their pathogenic potential and contributing to the bulk and structural integrity of the matrix.19 For the dental application, a “portable plasma sterilizer” (Ref. 20) is redesigned into a pen-size,21 that fits in a mouth and generates a cold air plasma jet. Emission spectroscopy of the plasma effluent shows that this cold air plasma jet still carries abundant RAO.21 In this work, an experimental study of the effects of this air plasma jet on oral pathogens and biofilms is reported. With air plasma, the treatment time is reduced to a few tens of seconds from a few minutes, which are normally needed with helium plasma treatment.22 The electric and emission characteristics and temperature measurements of a pen-size air plasma jet are presented in Sec. II. The sample preparation and experimental methods are described in Sec. III. The experimental results are presented in Sec. IV, showing plasma induced zone of growth inhibition in all six oral microorganism samples as well as

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FIG. 1. A schematic and photo of the plasma jet generator.

demonstrating a procedure of applying plasma treatment to prevent biofilm formation as a promising therapeutic approach to disinfect the cariogenic streptococci. To make sure that the plasma-assisted cell inactivation is not a nonculturablebut-viable state, fluorescence microscopy stained with LIVEDEAD bacLight bacterial viability has been performed on the control and treated biofilms for a comparison. Summary and discussions of the work are given in Sec. V. II. AIR PLASMA JET

This plasma jet generator consists of a pair of concentric electrodes, a ring-shaped permanent magnet, and a blower.20 A nozzle is introduced to direct the flow of the plasma effluent as well as to cover the electrodes for safety, so that the high voltage (HV) central electrode is not exposed. The nozzle opening (11 mm diameter) is positioned about 12 mm above the ring-shaped outer electrode. A schematic and a photo of the device are presented in Figs. 1(a) and 1(b), respectively. The ring magnet (12 (od)  6 (id)  2 (h) mm) produces a magnetic field of 0.1 T (Tesla) at its center. The central electrode, a cylindrical copper rod with a diameter of 3.175 mm, is inserted through this magnet and tight fit with a position holder, which keeps the central electrode along the central axis of the cylindrical frame of the plasma jet device. The magnet is placed right below the ring shaped outer electrode, which has inner and outer diameters of 4.7625 and 15.875 mm, respectively. Plasma is generated by 60 Hz arc discharges. The arc is diffused by injected airflow, from a blower

Phys. Plasmas 18, 073503 (2011)

connected to a tubular frame of the plasma device through a flexible tube, and by rotating around the central electrode through the interaction with the imposed magnetic field of the magnet. Specifically, the discharge current is pushed outward by the airflow to elongate the arc loop axially, in which electrons are energized by the electric field to produce free radicals; moreover, the arc loop is rotated azimuthally by the magnetic field of the magnet to avoid the formation of hot spots on the electrodes. The airflow speed at the nozzle exit of the device was measured by an air velocity meter (tsi model 1650). The airflow rate was then estimated from integrating the speed distribution over the cross section of the nozzle exit. The airflow rate and the average flow speed at the electrode gap (and nozzle exit) were evaluated to be about 1.74 l=s and 360 m=s (18 m=s), respectively. Such arrangements keep the discharge stable, spout the plasma effluent out of the nozzle by about 25 mm, uphold the generated nonequilibrium plasma effluent at low temperature, and reduce the erosion of the electrodes by the discharges. The plasma jet was run in periodic mode with a duty cycle of about 30%. A. Electric characteristics

The time varying voltage V(t) and current I(t) of the discharge were measured using a digital oscilloscope (Tektronix TDS3012 DPO 100 MHz and 1.25 GS=s), where V is the voltage of the central electrode of the plasma jet (the outer electrode is grounded). Current I was measured by a current loop (0.1V=1A rating) around the electric wire connected to the outer electrode. All of the discharge current passes through that wire. The product of the V and I functions gives the instantaneous power function P(t). These three time functions, V, I, and P, in one cycle (17 ms), are presented together in Fig. 2(a) to show their phase relationship. In the power supply, a capacitor and a diode are used for voltage doubling purpose. Thus, in each cycle of the 60 Hz AC input there is only one discharge occuring in the negative half cycle. The peak power in the discharge is less than 1 kW and the average power is about 135 W. The negative V-I characteristic of the discharge in a cycle, presented in Fig. 2(b), indicates that the discharges are in arc mode; however, the arc is prevented from constriction by the introduced airflow and magnetic field as explained previously.

FIG. 2. (Color online) (a) Voltage (V), current (I), and power (P) functions of the discharge in one cycle and (b) V-I characteristic of the discharge.

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Air plasma effect on dental disinfection

Phys. Plasmas 18, 073503 (2011)

FIG. 3. (Color online) (a) Axial temperature distribution of the plasma jet and (b) the surface temperature of an agar plate vs the exposure time to the plasma jet at 30 mm away; the plasma jet was run periodically, with 5-s on=10-s off.

B. Thermal temperature measurements

The temperature of the plasma effluent outside the nozzle was measured along the axis by a temperature meter “Omega DP460” (Omega Engineering, Inc., Stamford, CT). The response time of the thermocouple of the meter is about 0.5 s. We exposed the probe to the torch continuously for 30 s at three locations, 25, 30, and 40 mm away from the nozzle exit, to obtain a steady state reading from the meter. As shown in Fig. 3(a), the temperature drops from 60C to 47  C. In the disinfection tests, 30 mm exposure distance would be chosen, and the temperature of 55  C needed to be reduced. Thus, we would adopt a periodic approach, running plasma jet 5-s on=10-s off; the measured temperature increase for the plasma jet run 1–6 times is presented in Fig. 3(b). As shown, with the same total exposure time of 30 s, this periodic approach reduces the temperature from 55 to 47  C. When the probe was covered by agar, which would be used for a sample holder, the temperature was further dropped from 47 to 37  C.

mm cut through the central axis of the jet image with a height zt ¼ 20 mm. Charge-coupled-device (CCD) array detector captured spectrally dispersed image of the irradiated slit image, which was then analyzed row by row to obtain individual spectral intensities I(z,k). Given the magnification factor s and the size of single pixel of 25 lm, I(z,k) corresponded to an average intensity radiated from a plasma cylinder 0.3 mm high and 0.1 mm wide cutting through the central axis of the jet. Each spectral frame was calibrated using a black body radiation source to eliminate the error induced by the grating position. We measure the intensity changes in the atomic oxygen (OI) lines at 202.640, 777.1944, and 770.675 nm, and nitric oxide (NO) bands with band heads at 237.02 and 247.87 nm, along the (z) axis of the jet, from z ¼ 3.375 to 29.625 mm, where z ¼ 0 is the tip of the central electrode. From Fig. 5 we observe that close to the electrode (z ¼ 0) the amount of OI is the highest. Then as we move away from the electrode, the amount of NO increases and the amount of OI decreases. D. Electron excited OH rotational temperature

C. Emission characteristics

Shown in Fig. 4 is a scheme of the imaging spectroscopy used to determine axial distribution of the emission of the plasma jet. Image of the plasma jet was projected to the entrance plane of an imaging spectrometer, with magnification s ¼ zi=zt ¼ 0.08, where the entrance slit with a height zi ¼ 1.6

The hydroxide (OH) rotational temperature (Trot) was determined from the OH band with a band head at 306.36 nm. Fig. 6 shows the spectrum of this OH band from the radiation of the plasma jet. Using the iso-intensity method outlined by Dieke and Crosswhite,23 we determine Trot by matching the intensities

FIG. 4. (Color online) Scheme of the imaging spectroscopy.

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Phys. Plasmas 18, 073503 (2011) TABLE I. Identified spectral line intensities. K

k (nm)

I

2 3 4 5 6 7 8

308.0231 307.8028 307.4369 307.2199 307.0478 306.9177 306.8277

290 157 176 342 284 221 268

figure we show Te as a function of z. We see that the temperature increases as moving away from the electrode. III. MATERIAL AND EXPERIMENTAL METHODS FIG. 5. (Color online) Spatial distribution of the emission intensities of the NO bands (237.02 and 247.87 nm) and OI lines (202.64, 770.68, 777.19 nm).

of two of the OH lines belonging to the same branch. Table I presents the identified spectral lines and intensities, where K is the angular momentum when the electronic spin is disregarded. In the R2 branch there are two pairs of lines R2,K, with K equal to 2 and 6, and 3 and 4, which have matched intensities. They are shown in Fig. 6 as R2,2, R2,3, R2,4, and R2,6. We then used Table VIII of (Ref. 23) to determine the average OH rotational temperature from the two matches. Trot determined from lines 2 and 6 is 818 K and from lines 3 and 4 is 658 K. Thus, the average rotational temperature of OH is 738 6 80 K. E. Electron excitation temperature

By assuming a Boltzmann distribution for the intensities (Iki) of different Cu excited state spectral lines having the same lower level energy and different threshold excitation energies (Ek), the electron excitation temperature (Te) can be determined from: Iki ¼ ðgk Aki =kki Þ expðEk =kB Te Þ

(1)

Here gk is the statistical weight of the upper level k, Aki is the transition probability, and kki is the wavelength. We determined the temperature for the case when the nozzle was removed from the torch and the copper electrodes were cleaned (no Cu2O) by employing Eq. (1), see Fig. 7. In the

FIG. 6. Spectra of the OH band with band head at 306.36 nm.

A. Study of the zone of Inhibition of microorganism by plasma treatment

The suspension of sterile sodium chloride (0.89% of NaCl solution) and the defined microorganism culture was inoculated in an agar plate. The concentration of microorganism in the cultivating medium was approximately 106 c.f.u.=ml to form continuous overlay of microorganism coats on the agar plate.24 Samples would be exposed to the plasma effluent with a periodic approach, run 5-s on=10-s off for four different numbers of times, 1, 2, 4, and 6. Thus, there would be four sets of results from four different accumulated treatment times of 5, 10, 20, and 30 s for a comparison. The surface temperature of the sample holder increases with the exposure time, up from room temperature (23  C) to about 37  C after 30 s accumulated exposure (though the jet temperature experienced by the sample may raised to 47  C). The airflow was used as negative control. After the plasma exposure, samples of different microorganisms and different treatment times were cultivated at the best condition for each microorganism to observe the dependency of the zone of growth inhibition on the total treatment time. Included in the investigation are the six oral microorganisms, Actinomyces naeslundii CDCA1916 (ATCC12104), Candida albicans NIH3172 (ATCC14053), Streptococcus gordonii NCTC7865 (ATCC10558), Streptococcus mutans UA159 (ATCC700610), Streptococcus oralis NCTC11427 (ATCC35037), and Streptococcus sanguinis DSS-10 (ATCC10556). Each test was repeated three times.

FIG. 7. Axial distribution of the electron excitation temperature.

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Air plasma effect on dental disinfection

Phys. Plasmas 18, 073503 (2011)

FIG. 8. (Color online) A flow chart for biofilm tests.

B. Study of the plasma treatment effect on biofilm formation

S. mutans UA159 were inoculated in the Tryptone-yeast extract broth containing 1% sucrose to form biofilms on saliva-coated hydroxyapatite discs placed in a vertical position (HAP ceramic – calcium hydroxyapatite, 0.500 diameter – Clarkson Calcium Phosphates, Williamsport, PA) in batch culture at 37  C and 10% CO2.25 Normally, it takes five days to continuously cultivate the sample with the Tryptone-yeast extract broth and 1% sucrose, where the culture medium is changed daily, to establish a mature biofilm (which has a thickness of about 200 lm) on a saliva-coated hydroxyapatite disc. Thus, in the study of the plasma treatment effect on biofilm formation, five day cultivation was applied. After 24 h of initial biofilm formation, the biofilms were treated twice daily with a total accumulated treatment time of 30 s until day 5. The air-flow only was used as negative control. The treatment with the periodic approach was to guarantee the low temperature on the biofilm surfaces and the treatment time used in the experiment was based on the zone of inhibition data indicating thorough disinfection in 30 s. After the treatment, the biofilms (n ¼ 12) were harvested for the analyses of (A) bacterial viability, (B) dry-weight, (C) polysaccha-

FIG. 9. (Color online) A photo of the experimental setup.

ride composition content by colorimetric assays, and (D) morphology, using environmental scanning electron microscopy (ESEM). A flow chart with the treatment plan is presented in Fig. 8. Fluorescence microscopy was also used to show live and dead bacteria after plasma exposure. In the control group, similar biofilms were treated using the same airflow from the unignited plasma jet generator for the same amount of exposure time. IV. RESULTS

The plasma jet was secured on a fixed vertical holder with its circular nozzle exit facing downward to a sample holder, which is made of agar placed inside a glass dish, at a distance of 3 cm. The density and flux of RAO that reach the surface of the sample holder are estimated from the emission spectroscopy to be about 106 cm3 and 4  109 cm2 s1. A photo of the experimental setup is presented in Fig. 9. A. Zone of inhibition

After cultivation, the central region of the treated sample where was exposed to the plasma effluent is transparent to the agar plate due to the absence of colony forming units (c.f.u.) of the microorganisms, whereas in each of the controls, microorganisms have grown everywhere in the surface. This is exemplified in Fig. 10 by the images of the (a) control and (b) treated S. mutans samples. A zone of growth inhibition is circled in Fig. 10(b) of the treated sample and the zone of inhibitory is weighed by the diameter (cm) of the circle. It was found that there is a dose-response relationship showing the increase of the diameter of the zone of

FIG. 10. (Color online) Comparison of the growth situations between (a) the control and (b) the treated sample. The circle in (b) spots the zone of growth inhibition.

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Phys. Plasmas 18, 073503 (2011)

TABLE II. Diameter (cm) of the zone of inhibitory versus the plasma treatment time. Treatment Microorganism Actinomyces naeslundii CDC A1916 Candida albicans NIH 3172 Streptococcus gordonii NCTC 7865 Streptococcus mutans UA159 Streptococcus oralis NCTC 11427 Streptococcus sanguinis DSS-10

Plasma 5s

Plasma 10s

Plasma 20s

Plasma 30s

Air flow (control) 30s

0 0 2.5 2.0 0 2.6

2.0 0.8 2.7 2.7 2.5 2.7

2.5 1.7 3.0 3.2 3.0 3.0

3.0 2.1 3.5 3.5 3.5 3.0

0 0 0 0 0 0

inhibition with the increase of the exposure time. Samples of six microorganisms were treated by plasma with the periodic approach at four different total exposure times: 5s, 10s, 20s, and 30s, and each test were repeated three times. The average diameters of measured zones of growth inhibition are summarized in Table II. B. Effects of plasma on biofilm formation

The biofilm formed on the surface of a saliva-coated hydroxyapatite disc becomes mature after 5 days of unperturbed formation. In this state, aggregation of microbial cells are bound together by an extracellular matrix of polysaccharide and protein and the microorganisms become able to tolerate antimicrobial challenges that normally eradicate free-floating individual cells.26 In this investigation, plasma treatment was introduced to disturb the biofilm formation. The treated biofilm and its control were analyzed (p < 0.05) to compare their bacterial viability, biomass, and accumulated amounts of polysaccharide. The comparison is presented in Fig. 11. As shown, the viable c.f.u. was reduced by about 8 orders of magnitude. Moreover, the dry-weight and the amount of insoluble polysaccharide were reduced by more than 80%. The results suggest that the daily treatment with plasma effluent can fully inhibit the biofilm formation on the saliva-coated hydroxyapatite disc. It also seems that one treatment can already slow down the biofilm forming considerably. Environmental scanning electron microscope (ESEM= EVO 50 Zeiss, Germany) was used to exam the morphologies of the treated biofilm and its control. The fresh biofilms were ana-

FIG. 11. (Color online) Changes of the composition and viability of the treated biofilm of Streptococcus mutans UA159 in reference to the control.

lyzed directly in the microscope, without any prepreparation in the specimens. The ESEM microscope is capable of imaging delicate hydrated specimens in high pressure, by maintaining a humid atmosphere to prevent water loss in the specimen. The ESEM images of (a) the control, treated with airflow, and (b) the treated biofilm are presented in Fig. 12 for a comparison. The image (b) indicates that biofilm formation was fully inhibited with the daily plasma treatment. The fluorescence microscopy images were done with planktonic cells treated with airflow and with plasma. The live=dead bacLight bacterial viability kit (Molecular Probes, INC. Oregon,) was used to stain the live and dead bacteria with green and red colors or with white spots in (c) and white and gray spots in (d) for black and white version, respectively. The results are presented in Figs. 12(c) and 12(d). V. SUMMARY AND DISCUSSIONS

Reactive oxygen species, when reacting with nucleic acids, lipids, proteins, and sugars, causes oxidation of lipids and reducing sugars and amino acids, resulting to protein oxidation and degradation.27 In addition, the presence of moisture from atmosphere, the hydroxyl-radical, OH, can also play a significant role by chemically attacking the outer structures of bacterial and fungal cells and cause lipid preoxidation resulting from the susceptibility of unsaturated fatty

FIG. 12. (Color online) The architecture and surface topography of the biofilms after 5 days of formation examined by environmental scanning electron microscope: (a) biofilm treated with air flow during the formation as a control; and (b) biofilm treated with the plasma effluent during the formation. Fluorescence microscopy stained with LIVE ¼ DEAD bacLight bacterial viability: (c) control and (d) treated biofilm (White and Gray spots in (d): dead cells; White spots in (c): live cells).

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acids to attacks by hydroxyl radicals. The presence of O2 in air plasmas leads to the generation of ozone (O3), which interferes with cellular respiration to give a strong bactericidal effect.28 The emission spectroscopy indicates that the plasma jet carries an abundance of RAO. These chemical reactions, involving RAO in the present work, can destroy just about all kinds of microorganisms,29 and are putative pathways responsible for inhibiting the biofilm formation. Experiments of plasma treatments on six defined microorganisms’ cultures as well as on biofilm formation were conducted. The results, summarized in Table II, show that the plasma jet creates a zone of microbial growth inhibition in each treated sample and the zone of inhibitory increases with the plasma treatment time. In fact, after 20s treatment, all zones of microbial growth inhibition expand to be larger than the entire region directly exposed to the plasma jet. It indicates that the plasma effluent of the jet attracts to microorganisms. It can be concluded that the bactericidal agent carried by the plasma jet is effective to disinfect the grampositive bacteria and fungi. The plasma treatments during 5 days of biofilm formation did affect the biomass quantity and the polysaccharides composition, and the viability of the microorganisms in the biofilm, as shown in Fig. 11. The structural analyses and comparison, presented in Figs. 12(a) and 12(b), demonstrated that plasma treatment fully inhibited the biofilm formation. The polysaccharide structures were hardly seen in Fig. 12(b). The viability analyses and comparison, presented in Figs. 12(c) and 12(d), and then demonstrated the feasibility of using plasma to disinfect biofilms; only dead cells could be seen in Fig. 12(d). The antimicrobial effect of the plasma effluent was possibly the reason for the inhibition of the biofilm formation. Finally, the experimental result shows that the plasma jet can in fact eradicate the microorganisms deep in a mature biofilm (5 day-old), which are usually cannot be eradicated by the traditional antimicrobial agents; it is likely because the plasma jet disrupts the polysaccharides from the biofilm matrix, exposing the microorganisms to the plasma effluent. ACKNOWLEDGEMENTS

The authors are grateful to Dr. T. Bromage, Department of Biomaterials and Biomimetic, NYU College of Dentistry,

Phys. Plasmas 18, 073503 (2011)

New York, NY for the assistance with the ESEM images. Work was supported by NYU-Poly seed funds. 1

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