Interdigitated silver-polymer-based antibacterial surface systemactivated by oligodynamic iontophoresis – An empiricalcharacterization study

Rohan A. Shirwaiker & Richard A. Wysk &

Subhashinie Kariyawasam & Robert C. Voigt &Hector Carrion & Harriet Black Nembhard

Published online: 6 August 2013# Springer Science+Business Media New York 2013

Abstract There is a pressing need to control the occurrencesof nosocomial infections due to their detrimental effects onpatient well-being and the rising treatment costs. To preventthe contact transmission of such infections via health-criticalsurfaces, a prophylactic surface system that consists of aninterdigitated array of oppositely charged silver electrodeswith polymer separations and utilizes oligodynamic ionto-phoresis has been recently developed. This paper presents asystematic study that empirically characterizes the effects ofthe surface system parameters on its antibacterial efficacy,and validates the system’s effectiveness. In the first part ofthe study, a fractional factorial design of experiments (DOE)was conducted to identify the statistically significant systemparameters. The data were used to develop a first-orderresponse surface model to predict the system’s antibacterialefficacy based on the input parameters. In the second part ofthe study, the effectiveness of the surface system was vali-dated by evaluating it against four bacterial species respon-sible for several nosocomial infections – Staphylococcus

aureus, Escherichia coli, Pseudomonas aeruginosa, andEnterococcus faecalis – alongside non-antibacterial polymer(acrylic) control surfaces. The system demonstrated statisti-cally significant efficacy against all four bacteria. The resultsindicate that given a constant total effective surface area, thesystem designed with micro-scale features (minimum featurewidth: 20 μm) and activated by 15 μA direct current willprovide the most effective antibacterial prophylaxis.

Keywords Silver . Ions .Low intensity direct electric current(LIDC) . Antibacterial surface

1 Introduction

Compromised immune status of patients coupled with thehigh prevalence of pathogens including drug-resistant bacteria(e.g., methicillin-resistant Staphylococcus aureus (MRSA)) inhealthcare environments has aggravated the problem of nos-ocomial infections. Such infections are a cause of globalconcern in that they have affected both developed andresource-poor countries. According to the Centers for DiseaseControl and Prevention (CDC), over 1.7 million people areaffected by nosocomial infections in the United States (US)alone (Pollack 2010). In addition to their undesirable healtheffects and potential mortality, the treatment of these infec-tions is estimated to result in healthcare expenditures of $28–45 billion each year in the US (Scott 2009). Hence, there issignificant motivation to prevent the occurrences of nosoco-mial infections.

Contaminated inanimate surfaces in hospitals have beenshown to be one of the major contributors to the transmissionof nosocomial infections. Touching such surfaces may leadto contamination of the hand and subsequent transfer to otherinanimate objects or directly to patients (O’Connell andHumphreys 2000; Hayden et al. 2008; Casey et al. 2010).

R. A. Shirwaiker (*) : R. A. WyskEdward P. Fitts Department of Industrial and Systems Engineering,North Carolina State University, 400 Daniels Hall,Raleigh, NC 27695-7906, USAe-mail: rashirwaiker@ncsu.edu

R. A. Shirwaiker :R. A. WyskJoint Department of Biomedical Engineering, University of NorthCarolina and North Carolina State University,Raleigh, NC 27695, USA

S. KariyawasamDepartment of Veterinary and Biomedical Sciences, AnimalDiagnostic Laboratory, The Pennsylvania State University,University Park, PA 16802, USA

R. C. Voigt :H. Carrion :H. B. NembhardDepartment of Industrial and Manufacturing Engineering, ThePennsylvania State University, University Park, PA 16802, USA

Biomed Microdevices (2014) 16:1–10DOI 10.1007/s10544-013-9800-x

One strategy to prevent indirect contact transmission ofinfections is to design contact and work surfaces inhealthcare settings (e.g., door knobs, instrument trays,stethoscope chest-piece) to be intrinsically antibacterial. To-wards this end, we have recently designed a prophylacticsurface system stimulated by low intensity direct electriccurrent (LIDC) that is capable of reducing indirect contacttransmission of pathogens while also restricting the concep-tion of antibacterial resistant species (Shirwaiker et al. 2011;Shirwaiker et al. 2013). The system’s surface consists of aninterdigitated array of silver anodes and cathodes, with eachelectrode pair separated by an electrically resistive insula-tion. The presence of any aqueous media on the surfaceinitiates oligodynamic iontophoresis, resulting in the contin-uous release of silver ions (Ag+) into the media. When theconcentration of Ag+ exceeds a certain threshold concentra-tion, the ions will interact with any pathogens present in themedia potentially inhibiting or killing them; this antibacterialinteraction at the source of contamination results in prophy-laxis. Amongst various oligodynamic ion species, Ag+ hasdemonstrated potency against a wide range of infectiousbacteria and their antibiotic resistant strains (Panácek et al.,2006; Ellis, 2007). In addition, occurrences of silver-resistant bacteria are expected to be rare owing to theirmultiple modes of antibacterial action (Perceival et al.,2005; Silver et al., 2006).

The antibacterial surface system configuration is shown inFig. 1. Two silver electrodes of opposite polarities separatedby a polymer partition – the minimum requirement foroligodynamic iontophoresis to occur – constitute a basicfunctional unit of the system. The entire functional area ofthe surface is comprised of multiple basic functional units.The actual number of basic functional units will depend ontheir designed width and the available effective area of thesurface. Within each unit, the electrodes are responsible for

the antibacterial action through the release of Ag+ while thepolymer partition provides the necessary electrical insulationbetween them. The scalloped cross-section profile also pro-vides multiple advantages. The polymer partitions provide adurable contact/resting surface while shielding the softerfunctional silver electrodes from mechanical damage. Theyalso prevent the possibility of a short circuit if an electricallyconducting solid object were to be in contact with the sys-tem. A liquid medium in contact with the surface, however,will still be able to connect across alternate electrodes andinitiate the necessary iontophoresis reaction. Based on theo-retical analysis, we identified five system parameters – phys-ical, electrical or biological in nature – that may have aprofound effect on the antibacterial performance of the sys-tem. These include: basic functional unit width (W),partition-electrode thickness ratio (R), current (I), voltage(V), and the type of bacteria (B) to which the surface isexposed.

The basic functional unit width (W) governs the resolu-tion of the surface in terms of the aqueous droplet size thatthe system can act upon, and the current density in theelectrodes. The partition-electrode thickness ratio (R) hasan effect on the hydrophobicity of the surface, especially atthe micro-scale, and also the system’s mechanical function-ality. Electric current (I) is a critical parameter because it isresponsible for the release and concentration of Ag+, asgoverned by Faraday’s first law of electrolysis. Voltage (V)applied across the electrodes in the system creates the elec-trical field that initiates electric current in the system. It isimportant to note that these four variables are the designparameters that can be directly controlled by the systemdesigner and manufacturer, and some of their interactionswhich are also critical to the system performance can beindirectly controlled by varying these primary parameters.Current density is one such electrochemical parameter which

Fig. 1 Concept of theinterdigitated silver-polymer-based antibacterial surfacesystem activated byoligodynamic iontophoresis

2 Biomed Microdevices (2014) 16:1–10

is governed by the current and the dimensions of the elec-trode. Given a constant system current (I) and constant totalvolume and contact area of a droplet, the current density willbe higher in the surface with smaller basic functional unitwidths (W). Based on fundamental electrochemistry princi-ples, higher current density leads to a faster rate of reactionand ion release at the anode (Zoski et al. 2007). Although thetype of bacteria (B) is not a design parameter of the systembut a non-controllable factor in the system’s functional en-vironment, it is known that Gram-positive and Gram-negative bacteria respond differently to antibacterial agents.Thickness of the peptidoglycan layer in Gram-positive bac-terial cell wall limits the penetration of Ag+ through the cellwall, and hence its antibacterial potency, as compared toGram-negative bacteria (Feng et al., 2000; Jung et al., 2008).

The importance and characteristics of these parametersare discussed in more detail elsewhere (Shirwaiker et al.,2011). The results of that previous study using micro-scalefeatured surfaces (minimum feature width = 20 μm) stimu-lated by 15 μA current had demonstrated, for the first time,the effectiveness of the system design independently againstboth Gram-positive (S. aureus, ATCC 29213) and Gram-negative (Escherichia coli, ATCC 25922) bacteria. In addi-tion, we have also confirmed the biocompatibility of thesurface system, making it favorable for application to med-ical device surfaces (Samberg et al., 2012).

The primary objective of the present study is to modelthe antibacterial behavior of the system based on the fivesystem parameters. We have used fractional factorial de-sign of experiments (DOE) to characterize the effect ofscaling the system design parameters (W, R, I , V), and thebacteria type (B) on the antibacterial efficacy (percentagereduction in bacteria) of the surface over a 30 min timeinterval. The fractional factorial experimental design wasused instead of a one factor at a time (OFAT) analysis toaccount for any interactions between the input parameters.In the second part of the study, we have evaluated thesystem’s effectiveness against S. aureus (ATCC 29213)and E. coli (ATCC 25922) which were used in the DOEand two additional bacteria – Pseudomonas aeruginosa(ATCC 27853), and Enterococcus faecalis (ATCC 29212) –alongside non-antibacterial control surfaces. Together, thefour reference strains of bacteria tested in this study and theirvariants account for a sizeable proportion of nosocomialinfections worldwide.

2 Experimental methods

2.1 Experimental design and statistical analyses

Because the goal of the first part of this study was to char-acterize the system rather than to optimize it, a replicated

25 −1 factorial experiment (resolution = 5) was used to obtaina first-order response surface model with main effects andinteraction terms using regression analysis. The five fac-tors (system parameters) and factor levels were chosenbased on the previous theoretical system design analysesand the preliminary experimental results (Shirwaikeret al., 2011). The factors and their levels are provided inTable 1.

For the basic functional unit width (W) factor, the ratio ofthe width of individual electrodes to the width of partitionwas held constant at 2:1 at both factor levels; functionalelectrodes accounted for 80 % of the surface area of eachbasic functional unit, irrespective of its absolute width. Asthe samples used in this study had an effective functionalarea of 10 mm×10 mm, each micro-scale and meso-scalesurface contained 100 and 5 basic functional units, respec-tively. A dedicated constant voltage power source was usedto achieve the two voltage factor (V) levels. External resistorcircuits were designed using standard breadboard and resis-tors (RadioShack, Fort Worth, TX) to generate the twodifferent current (I) levels at the two voltage levels. Fourunique circuits were built such that each current level couldbe obtained at each constant voltage level. Since the resis-tance of the external circuit to obtain the desired micro-current levels was significantly higher than inherent resis-tance of the aqueous media, any reasonable variations inresistivity of the media itself were not expected to affectthe system current and performance. S. aureus and E. coliwere used in the DOE as the representative species for Gram-positive and Gram-negative bacteria, respectively. Tempera-ture and humidity, which are two non-controllable factors inthe system’s working environ that can affect its performance,were held constant in this experimental design at 37 °C(±1.5 °C) and 70 % (±15 %), respectively. While 37 °C isthe ideal growth temperature for most bacteria, the highhumidity prevents evaporation of the biomedia droplet dur-ing the testing interval. Thus, these two conditions ensuredthat the system was evaluated for antibacterial efficacy underoptimal growth conditions for the pathogens – a worst-

Table 1 Factors and factor levels used in the 25−1 experimental design

Low-level High-level Factor Type

Basic functional unitwidth (W) (μm)

100(micro-scale)

2,000(meso-scale)

Quantitative

Partition-electrodethickness ratio (R)

1.1 1.5 Quantitative

Electric current(I) (μA)

1.5 15 Quantitative

Voltage (V) (V) 6 15 Quantitative

Type of bacteria (B) Gram-negative(E. coli)

Gram-positive(S. aureus)

Qualitative

Biomed Microdevices (2014) 16:1–10 3

case working environment scenario from an application per-spective. Although the specific temperatures of the surfaceswere not monitored during testing, preliminary experimentshad confirmed that there was not noticeable surface heatingdue to the current over the testing interval which could affectthe system behavior. The percentage change in concentrationof bacteria (cfu/ml) on the surface over the 30 min timeinterval was used as the response variable (Eq. 1).

Percentagereduction inbacteria

¼ Starting concentration−Final concentrationStarting concentration

� �� 100

ð1Þ

MINITAB 16 (Minitab, State College, PA) statisticalsoftware package was used for the factorial design andstatistical analyses. A completely randomized 25−1 designmatrix with two replicates was generated; the principalfraction was used, and the 32 total runs (16 runs for eachreplicate) were randomized using 1 as the base generator.All the surface testing experiments were performed in thisrandomized order over a 21-day period. Based on theexperimental results, an initial model of the system wasformed. Analysis of variance (ANOVA) was performed toidentify and eliminate any non-significant factors and in-teractions. Finally, the revised first-order response surfacemodel to predict the system performance was obtained andanalyzed to verify adequacy.

2.2 Fabrication of test surface samples

Because of the order of magnitude difference between basicfunctional unit widths (W) at the two factor levels, twodifferent sets of processes were used to fabricate the micro-scale (W=100 μm; electrode width: 40 μm, partition width:20 μm) and meso-scale (W=2 mm; electrode width: 800 μm,partition width: 400 μm) test samples. The process plan formicro-scale sample fabrication that consists of two sets ofcontact lithography is summarized here. Four test samples(functional area 10 mm×10 mm) were fabricated on eachsilicon substrate coated with 120 nm SiO2 on both sides(Ø100 mm, Silicon Quest International Inc., Reno, NV). Tobegin, the substrate was thoroughly cleaned usingNanostrip® (OM Group Inc., Cleveland, OH), acetone,isopropanol (IPA) and deionized (DI) water, and blow-dried with N2 gas. Then, AZ 5214 E image reversal photo-resist (Clariant, Charlotte, NC) was spin coated onto thesubstrate (thickness = ~ 1.5 μm) and contact lithographywas performed to create channels for silver electrode depo-sition. A 1.2 μm thin film of silver (99.95 %, Kurt J. LeskerCo., Clairton, PA) was deposited on top of a 50 nm seed layerof chromium (99.95 %, Kurt J. Lesker Co., Clairton, PA) on

the substrate using electron beam physical vapor deposition(EB-PVD) (Denton Vacuum Systems Inc., Moorestown,NJ). This film covered the empty channels on the substratesas well as the top sacrificial photoresist layer. Followingdeposition, lift-off was achieved by cleaning the substratein NANO® Remover PG (MicroChem Corp., Newton, MA)to leave behind the patterned silver electrode structures. Inthe subsequent steps, another contact lithography operationwas performed to create the NANO® SU-8 2 (MicroChemCorp., Newton, MA) partition features of the desired thick-ness between the interdigitated silver electrodes. Finally,each silicon substrate was cleaved into the four individualtest samples using a diamond tip cutter. Two electrical wires(22 AWG, RadioShack, Fort Worth, TX) were soldered tothe ends of two electrodes of opposite polarities for eachsample. This process plan is described in greater detail else-where (Shirwaiker et al. 2011).

Meso-scale test samples were fabricated using conven-tional manufacturing processes on Acrylite® FF Acrylic(US Plastic Corp., Lima, OH) substrates sectioned intosmaller coupons. After cleaning with IPA, 800 μm wideslots with 400 μm separations in between were end milledinto each sample to create channels for the electrode array(functional area: 10 mm x 10 mm). A film of silver(99.99 %, SPI supplies, West Chester, PA) was coatedonto each coupon followed by drying at room temperaturefor 30 min, and a 1 h baking cycle in a convection oven at60 °C to allow the solvents to evaporate. In the finalfabrication step, the top surface of each sample was facemilled to obtain them in their final form with acrylicpartitions between silver electrodes. Two electrical wires(22 AWG, RadioShack, Fort Worth, TX) were embeddedinto the ends of two electrodes of opposite polarities andsecured using conductive paint.

All samples were subjected to metrology analyses (e.g.optical microscopy, surface profilometry etc.) and current–voltage (I-V) characterization to confirm their final featuredimensions and electrical characteristics. Non-antibacterialcontrol samples for the second part of the study were cut outof Acrylite® FF Acrylic (US Plastic Corp., Lima, OH) sub-strates and cleaned with IPA. Finally, all samples were storedindividually in sterile plastic containers to prevent contami-nation prior to the experiments.

2.3 Antibacterial efficacy testing

The antibacterial efficacy testing methodology described herewas developed to evaluate test and control samples in anenvironmentally controlled chamber against both Gram-positive and Gram-negative bacteria. The temperature andhumidity inside the testing chamber were controlled at 37 °C(±1.5 °C) and 70% (±15%) andmonitored continuously usinga thermometer and hygrometer. At the beginning of each

4 Biomed Microdevices (2014) 16:1–10

experimental run, the chamber was thoroughly cleaned usingIPA. Petri dishes cleaned with IPA and sterilized under ultra-violet (UV) light for 1 h were placed into the chamber to holdthe samples. All samples to be evaluated during the experi-mental run were also disinfected under UV light for 1 h andplaced inside the sterile petri dishes. The test surfaces wereconnected to the appropriate electrical circuit and powersource located externally.

Bacteria cultures – S. aureus (ATCC 29213) and E. coli(ATCC 25922) for the first part and additionally, Pseudomo-nas aeruginosa (ATCC 27853), and Enterococcus faecalis(ATCC 29212) for the second part of the study –were freshlygrown in 4 ml of tryptic soy broth (TSB) incubated at 37 °Cfor 15 h before each testing run. Unless otherwise stated, allmedia used in these microbiological experiments wereobtained from Remel (Lenexa, KS). After 15 h of incubation,five 1:10 serial log dilutions were performed by adding 45 μlof inoculant from the previous log dilution (or starting stockfor the first dilution) to 405 μl of fresh TSB in a 2 ml culturetube. Each culture tube was vortexed for 15 s before prepar-ing the next dilution in order to ensure homogeneity. 33 μlvolume droplets from the fifth log dilution were used for theactual testing experiments, as this was determined to be themaximum droplet volume that could be safely sustained onthe test sample without flowing outside the effective function-al surface envelope or evaporating within the 30 min testinginterval. To determine the starting concentration of this inoc-ulant, 33 μl of the fifth log dilution was inoculated into1,020 μl of phosphate buffered saline (PBS, pH 7.4) solutionand vortexed for 1 min. A 200 μl volume of this solution wasplated onto a growth media plate (5 % sheep blood agar for S.aureus and E. faecalis, andMacConkey agar for E. coli and P.aeruginosa) and incubated at 37 °C for 21 h.

To test the samples, the fifth log dilution culture tube wasvortexed for 15 s and a 33 μl droplet was pipetted onto eachsample from a height of approximately 5 mm. During thesecond part of the study, test samples were inoculated before

the control samples, and the power source was turned on assoon as the last control sample was inoculated. The setup wasleft undisturbed for the 30min test interval after which, the testsamples were electrically disconnected. After 30 min, allsamples were picked up with sterile forceps in the same orderthat they were inoculated, and immersed into individual san-itized Sterilite containers with 1,020 μl PBS. The containerswere then vortexed for 1 min to wash the inoculant off thesample surfaces and into the PBS solution. Similar to theprocedure to calculate the starting colony count at the begin-ning of the testing experiment, 200 μl of this solution wasplated onto a growthmedia plate and incubated at 37 °C. After

Fig. 2 Examples of test samples in comparison to a US dime: (a)Micro-sample, and (b) meso-scale sample. These were used for theantibacterial efficacy characterization and validation experiments

Table 2 Randomized 25−1 design matrix and experimental results

Std Order Run Order W I V R B Averagepercentagereductiona

2 1 2,000 1.5 6 1.1 GN 66.40

26 2 2,000 1.5 6 1.5 GP −0.59

22 3 2,000 1.5 15 1.1 GP −1.05

1 4 100 1.5 6 1.1 GP 5.23

15 5 100 15 15 1.5 GN 100

24 6 2,000 15 15 1.1 GN 90.96

6 7 2,000 1.5 15 1.1 GP −8.33

20 8 2,000 15 6 1.1 GP −0.36

9 9 100 1.5 6 1.5 GN 100

12 10 2,000 15 6 1.5 GN 97.92

16 11 2,000 15 15 1.5 GP 1.76

3 12 100 15 6 1.1 GN 99.63

5 13 100 1.5 15 1.1 GN 100

32 14 2,000 15 15 1.5 GP 0.50

4 15 2,000 15 6 1.1 GP 0.47

13 16 100 1.5 15 1.5 GP 3.74

28 17 2,000 15 6 1.5 GN 93.56

27 18 100 15 6 1.5 GP 14.26

14 19 2,000 1.5 15 1.5 GN 70.23

18 20 2,000 1.5 6 1.1 GN 75.78

17 21 100 1.5 6 1.1 GP 8.57

8 22 2,000 15 15 1.1 GN 95.74

31 23 100 15 15 1.5 GN 100

11 24 100 15 6 1.5 GP 18.89

10 25 2,000 1.5 6 1.5 GP −5.61

29 26 100 1.5 15 1.5 GP 11.76

23 27 100 15 15 1.1 GP 16.86

30 28 2,000 1.5 15 1.5 GN 61.22

25 29 100 1.5 6 1.5 GN 100

19 30 100 15 6 1.1 GN 100

7 31 100 15 15 1.1 GP 21.23

21 32 100 1.5 15 1.1 GN 100

a Negative sign indicates increase in bacteria concentration over the30 min time interval

Biomed Microdevices (2014) 16:1–10 5

21 h of incubation, colony counts were recorded from allplates and calculations were made to determine the viablecolony forming units/ml (cfu/ml) and percentage change inconcentration from all surfaces after the testing interval.

3 Results

Examples of a micro- and a meso-scale sample fabricatedusing the fabrication methods described in Section 2.2 andused for the antibacterial efficacy characterization and vali-dation experiments are shown in Fig. 2.

The randomized design matrix and experimental results arepresented in Table 2. The result of the 28th experimental runwas

identified as an outlier. The main effects and interaction plots forthe response variable, and the normal plot for standardizedeffects (α=0.05) are shown in Fig. 3a, c. From these plots, themain effects ofW, I and B, and the interactions between V-R, I-B, and W-I were noted to have a discernible effect on thepercentage reduction in bacteria. Results from ANOVA werealso consistent with these plots (α=0.05; p-values for W=0,I=0, B=0,W-I=0.001, I-B=0.043, and V-R=0).

Two factors with insignificant main effects (V, R) wereretained for further analyses only because their 2-way inter-action was significant (p-value=0), and the model was re-vised by eliminating all insignificant 2-way interactions. Therevised Minitab output of ANOVA in coded units and esti-mated coefficients in uncoded units is presented below.

Analysis of Variance for % Reduction (coded units)

Source DF Seq SS Adj SS Adj MS F P

Main Effects 5 61131.1 61131.1 12226.2 996.77 0.000

W 1 2138.5 2138.5 2138.5 174.34 0.000

I 1 841.3 841.3 841.3 68.59 0.000

V 1 2.8 2.8 2.8 0.23 0.635

R 1 0.4 0.4 0.4 0.03 0.862

B 1 58148.1 58148.1 58148.1 4740.66 0.000

2-Way Interactions 3 785.3 785.3 261.8 21.34 0.000

W * I 1 204.7 204.7 204.7 16.69 0.000

I * B 1 61.3 61.3 61.3 5.00 0.035

V * R 1 519.3 519.3 519.3 42.33 0.000

Residual Error 23 282.1 282.1 12.3

Lack of Fit 7 78.1 78.1 11.2 0.88 0.546

Pure Error 16 204.0 204.0 12.7

Total 31 62198.5

Unusual Observations for % Reduction

Obs StdOrder %Reduction Fit SE Fit Residual St Resid

28 30 61.217 69.064 1.857 −7.847 −2.64R

R denotes an observation with a large standardizedresidual.

Estimated Coefficients for % Reduction using datain uncoded units

Term Coef

Constant −5.4186

W −0.0118591

I 0.345459

V 5.75243

R 46.4511

B −40.9361

W * I 0.000394433

I * B −0.205050

V * R −4.47589

DF Degrees of freedom, Adj SS Adjusted sum of squares, F Fisher statistic, Seq SS Sequential sum of squares, Adj MS Adjusted mean of squares,P p-value

6 Biomed Microdevices (2014) 16:1–10

The normal probability plot of residuals (Fig. 4) and graphs ofresiduals versus fitted values and residuals versus order of data

confirmed all standard assumptions regarding validity of therevised model – the residuals are (1) randomly and normally

W V

R

I

B

W

I

V

R

B

I

WI

IBVR

W

B

(a)

(b)

(c)

Fig. 3 Plots of (a) Main effectsand (b) Interactions for theresponse variable, and (c)Normal plot of standardizedeffects

Biomed Microdevices (2014) 16:1–10 7

distributed, (2) do not correlate with the predicted responsevalues, and (3) do not exhibit any trends over time. Based onthe complete statistical analyses, the first-order response sur-face model with main effects and interaction terms usingregression analysis is given by Eq. 2.

Percentagereduction inbacteria

¼ −5:4186− 0:0119�Wð Þ þ 0:3455� Ið Þþ 5:7524� Vð Þ þ 46:4511� Rð Þ þ 40:9361� Bð Þþ 0:0004�W � Ið Þ− 0:2051� I � Bð Þ− 4:4759� V � Rð Þ

ð2Þ

Results of the second part of the study in which the micro-and meso-scale test samples alongside non-antibacterial con-trol surfaces were evaluated against S. aureus, E. coli, E.faecalis, and P. aeruginosa are presented in Table 3. Theblood agar and MacConkey agar plates from the testing

against S. aureus and E. coli are presented in Fig. 5a and b,respectively.

4 Discussion

The Pareto chart (Fig. 6) highlights the relative contributionsof the standardized effects of the parameters to the responsevariable. The model in uncoded units (Eq. 2) obtained fromthe first part of the study (DOE) characterizes the system’santibacterial behavior. By analyzing the Pareto chart andcoefficients of main effects terms in the model, inferencescan be made about how the system input parameters affectthe system’s ability to inhibit bacteria.

B has the most significant effect on the system’s perfor-mance. Note that the input in the model for B is a coded unit(−1 for Gram-negative and +1 for Gram-positive) since it is a

Fig. 4 Normal probability plotof residuals for the revised model

Table 3 Antibacterial efficacy testing results using micro and meso-scale test and acrylic control samples against S. aureus, E. coli, E.faecalis and P. aeruginosa over a 30 min interval. Test samples wereactivated by 15 V–15 μA power circuit

Percentage reduction in bacteriaa

Micro-scale sample Meso-scale sample Controlsample

S. aureus 17.72 8.23 −10.13

E. coli 100 84.21 −13.16

E. faecalis 12.71 3.39 −6.78

P. aeruginosa 75 39.13 −8.70

a Negative sign indicates increase in bacteria concentration over the30 min time interval

Micro-scale sample Meso-scale sample Control sample

(a)

(b)

Fig. 5 Growth media plates from the antibacterial efficacy validationexperiments: (a) Blood agar plates from testing against S. aureus, and(b) MacConkey agar plates from testing against E. coli

8 Biomed Microdevices (2014) 16:1–10

qualitative factor. The negative coefficient of B indicates thathigher reduction is observed in case of Gram-negative bac-teria. This aspect of the system’s behavior is in accordancewith other results from literature; Gram-positive bacteriapossess a stronger defense mechanism against silver. Interms of the actual design parameters of the system, W hasthe most substantial impact on its antibacterial efficacy. Thenegative coefficient of W indicates that a decrease in thesystems basic functional width results in an increase inreduction of bacteria. This also conforms to the theoreticalhypothesis that smaller electrode sizes are responsible forhigher current densities (current I remaining constant)resulting in faster rate of reaction, more uniform diffusion ofAg+ throughout the droplet, and hence better antibacterialefficacy. The positive coefficient of I indicates a directlyproportional relationship between the system current and itsantibacterial performance. This is in agreement with the prin-ciple of oligodynamic iontophoresis (governed by Faraday’sfirst law) according to which, higher current leads to higherconcentrations of Ag+ and hence, higher reduction in bacteria.Although the voltage and thickness ratio terms do not con-tribute significantly to the model, their positive coefficientsindicate that higher values of these factors may be beneficialfor the system’s performance. It is difficult to analyze thetrends with the interaction terms since they do hold anyphysical value or significance.

The results of the second part of the study also validatesome of these inferences from the system model. For all fourbacteria species, the micro-scale surface (electrode width:40 μm; partition width: 20 μm) samples demonstrated betterantibacterial efficacy than themeso-scale samples. In addition,the antibacterial efficacy was significantly higher againstGram-negative bacteria (E. coli and P. aeruginosa) than theGram-positive ones (S. aureus and E. faecalis). Based onFaraday’s first law, the concentration of Ag+ under idealconditions in a 33 μl aqueous droplet after 30 min of testingwould be approximately 1 ppmwith a system current of 15 μA

and approximately 0.1 ppm with a system current of 1.5 μA.The results (antibacterial efficacy) of this study are inagreement with other studies from literature which re-ported reduction in S. aureus and E. coli at similar Ag+

concentrations (Jung et al., 2008).Most importantly, in all the tests in this study, the

antibacterial surface system performed much more favorablythan the acrylic control surfaces. This is critical from anapplications perspective and iterates the need to furthertranslate the interdigitated silver-polymer-based antibacterialsurface system design to health-critical devices and surfaces.

5 Conclusion

Although antibacterial potency of electrically activated Ag+

has been reported in literature for over two decades, we haverecently, for the first time, configured and implemented thisconcept for surface system applications. Such surfaces canprimarily be used in healthcare environments for touch-contact and work surfaces (e.g., stethoscope chest-piece,door knobs, instrument trays etc.) to prevent indirect contacttransmission of nosocomial infections. In this study, we haveconducted an empirical characterization of the surface systemdesign to identify critical parameters associated with the re-lease and oligodynamicity of Ag+, and model how these affectits antibacterial efficacy. We have also validated its efficacyagainst four nosocomial infection-causing pathogens. Thesystem was more effective against Gram-negative bacteria asanticipated. Further, the results demonstrate the importance ofmicrotechnology to this surface system. In terms of the actualdesign parameters of the system, the width of the functionalfeatures has the most significant effect on its antibacterialefficacy; scaling them down significantly enhances systemperformance. We believe that the results of this study andthe ensuing empirical first-order response surface model willaid further understanding of the surface system and assist inoptimization studies in the future.

Acknowledgments This work was supported in part by a researchgrant from the Penn State Clinical and Translational Sciences Institute(CTSI). The authors acknowledge the use of facilities at the PSU site ofthe NSF NNIN under agreement # 0335765 for the micro-scale proto-type fabrication, and the Animal Diagnostic Lab for the microbiologicaltesting. The authors also thank Dr. Douglas Wolfe and Mr. ThomasMedill of the Advanced Coatings Lab in the Applied Research Labo-ratory (ARL) at Penn State for their help with silver thin film deposition.

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Fig. 6 Pareto chart of the standardized effects in the revised model

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