Electrochemical measurements of biofilm development using polypyrrole enhanced flexible sensors

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Abstract

Bacterial biofilms can be beneficial or detrimental, and are capable of forming on virtually any surface. There is a great need for an in situ sensor able to detect and characterize the developmental stages of bacterial biofilms. We have developed an electrochemical approach to detect and characterize bacterial biofilms using polypyrrole (PPy) enhanced flexible biofilm sensors based on organic substrates of Polyethylene terephthalate (PET). PPy films act as a functionalization material on gold electrodes to reduce their electrical impedance thereby enhancing the detection of electrochemical signals. Flexible PET substrates enable sensors to be placed in systems with complex geometries and to be produced using low cost roll-to-roll manufacturing. Measurements of electrochemical impedance spectroscopy (EIS) using the PET flexible biofilm sensors were correlated with fluorescence microscopy using bacteria that express green fluorescent protein (GFP). The biofilm sensors successfully detected the changes of charge transfer resistance and capacitance corresponding to the maturing stages of biofilm development. The charge transfer resistance increases during the early stages of biofilm maturation and decreases in the later stages of development.

Introduction

Bacteria exist both as planktonic (free swimming) and as biofilms (attached to surfaces) [1]. Bacterial biofilms are detrimental in a large number of environments ranging from wound beds causing chronic infections [2], [3], [4] to pipes causing clogging and corrosion [5], [6], [7], [8]. Bacteria within biofilms are protected by a viscous matrix, known as EPS (extracellular polymeric substances), mainly composed by polysaccharide, resulting in 50–1000 times higher antibiotic resistance [2], [9]. The EPS is porous allowing the exchange of nutrients and the elimination of waste [10], [11]. Biofilms are one of the major causes of microbiologically influenced corrosion (MIC) shortening the service time of pipes, of increased health risks due to contaminated municipal water, and of contamination in the food and pharmaceutical industries [5], [6], [7], [8]. One of the common targets of biofilm MIC is cast iron pipes, but even stainless steel is not free of biofilm MIC [5], [6], [7]. The growth of microbial biofilms on a metal surface produces an open circuit potential (OCP) or free corrosion potential [6], [7].

One of the emerging methods for biofilm detection is the use of electrochemical measurements. Biofilms are immobilized on electrodes to generate electrochemical signals captured by a potentiostat system [7], [12], [13], [14], [15], [16], [17]. A common method utilizes coupons as working electrodes [7], [13]. A coupon is usually the size of a coin and is connected to a potentiostat as a working electrode. The counter and reference electrodes are submerged into the media while conducting the electrochemical measurement. To prevent biofilm growth on these electrodes, they are removed between measurements. Each working electrode coupon can be taken out for microscopy observations, for sampling the biofilm to perform colony-forming unit (CFU) counting, and for evaluating MIC. An advantage of the coupon method is that the reference electrode is maintained free of the biofilm. However it is difficult for a coupon-based system to conform to the geometry of a wound bed for in vivo applications or the internal surface of a pipe.

One advantage of the EIS approach is that the data can be used to develop model circuits that lead to a better understanding of the measurements, and can also be used to model the various stages of biofilm development. The latter can be used to improve our knowledge of biofilm development, and to use EIS as a predictor of biofilm presence and development for field monitoring applications. There are many equivalent circuits for modeling the electrolyte/biofilm/electrode interface from EIS measurements, but the Randles circuit and its derivatives are among the most widely-used models [12], [13], [14], [15], [16], [17]. The key parameters in Randles circuits include: double layer capacitance (Cdl) or constant phase element (CPE) impedance (ZCPE), Warburg impedance or diffusion-limit resistance (Zw), and charge-transfer resistance (Rct). Additional components are integrated to the Randles circuit forming a more complex circuit to improve the modeling of the EIS data. These components include biomaterial capacitance (Cb) and biomaterial resistance (Rb) [14]. Dheilly et al. [13] used EIS to monitor cell attachment and biofilm maturation. Increased cell population during the attachment stage and increased biofilm surface coverage resulted in higher charge-transfer resistance. Ben-Yoav et al. [14] investigated the development of Escherichia coli biofilms on ITO (indium tin oxide) electrodes. The biomaterial resistance decreased during the cell attachment stage, but increased throughout the later stages of development. These authors also claimed that the different parameters of an optimized equivalent circuit may be due to the different stages of biofilm maturation. Kim et al. [15] used platinum disks as electrodes to examine the influence of bacteria and biofilms on the change of the double layer capacitance. Munoz-Berbel et al. [16] examined the influence of biofilm formation in a drinking water system using on-chip gold electrodes. Both of these studies [15], [16] found that the electrically insulating biofilm fouling on the working electrode decreased its effective area, resulting in a reduction in capacitance. Xu et al. [17] monitored marine biofilm adhesion on graphite electrodes showing an increase in the capacitance and decreased charge transfer resistance because the biofilms were electrochemically active.

Improving the function of electrochemical electrodes and their integration with the biological system under study is an ongoing and important effort. One approach is to use conductive polypyrrole (PPy) based films electrochemically deposited on the electrodes [18], [19], [20]. The conductivity of PPy films depends on its oxidation state that is determined by the electrolysis conditions, such as deposition potential, current density, and the concentration of solvent and dopant [19], [21]. PPy film coated electrodes have good biocompatibility in vivo compared to bare metal electrodes [22], [23]. In addition, the selectivity of the adsorption of target proteins can be manipulated with various coupling dopants [24]. Another attractiveness of PPy films for biosensors is its long term-environmental stability [18], [25].

This study introduces PPy functionalized flexible, electrochemical sensors to monitor real-time biofilm growth on microelectrodes as shown in Fig. 1(a). Flexible biofilm sensors have distinct advantages such as optimal geometric conformation for specific applications and low cost. A flexible sensor allows conformal fitting on the irregular surface of a wound bed and to the inside surfaces of water pipes in a wide variety of industrial and infrastructural applications. Therefore it is possible to embed such a biosensor into a “smart bandage” to detect biofilms and to identify pathogenic species. The flexible biosensor can be rolled into a cylinder and fit into a pipe without blocking the flow while maintaining the same shear force on the sensor's surface for biofilm development as on the pipe's surface. Flexible electronics can be fabricated by the roll-to-roll method that is ultra-low cost. Microelectrodes provide 2 major advantages: operation in highly resistive solution with no need for supporting electrolyte and high sensitivity compared to macroelectrodes. Our motivation to use PPy for functionalizing the working electrodes is twofold: (1) PPy is a conductive polymer that can serve as a catalytic electrode material to produce a low impedance sensing system for real-time EIS measurement without adding a redox agent; (2) the surface roughness of PPy is more adhesive to bacteria which enhances biofilm immobilization on the surface of the working electrodes. The biofilm sensor used in this study is composed of a 2-electrode system, as opposed to commonly used 3-electrode systems. The 2-electrode system was chosen due to the ease of embedding it into a well-designed electric impedance meter for in-field applications. The objective of the current study is to demonstrate the capability to electrochemically sense biofilms, specifically, the feasibility of sensor recognition on flexible substrates, the assessment of sensitivity and the practical use of PPy as a functional material. This is a necessary step toward producing a low-cost, biofilm sensor for in-line and real-time biofilm monitoring. Future work will further delve into electrode pad placement, and compensation for geometry of the sensing surfaces during monitoring.

Section snippets

Design and fabrication of flexible biosensor

The sensor design allows optimization of the electrochemical measurement by varying the size ratio between the reference electrode(s) and the working electrodes, the distance between a reference electrode and a working electrode, and the sensitivity based on the size of the working electrodes. The electrode layout is shown in Fig. 1(b). It contains 3 larger electrodes that can be used individually or in combination as reference electrodes. The largest one in the middle of the electrode array is

Results

The EIS measurements and fluorescent microscopy observations were recorded using the PPy coated 150 × 150 μm working electrodes on 3 PPy sensors. Representative EIS data, the charge transfer resistance (Rct) and the exponent of adsorption CPE impedance (n), and the fluorescent microscopy observations are shown in Fig. 3, Fig. 4, Fig. 5, Fig. 6. The improved response of the PPy sensors was demonstrated by comparison to a bare Au sensor (Fig. 7).

The fluorescent images in Fig. 3 demonstrate biofilm

Discussion

In this study, biofilm development stages were determined by analyzing fluorescence images that were subsequently correlated with the EIS data. Biofilms of P. aeruginosa are considered to be an electrically inactive material [13], [15], [16], [26]. The variation of the Rct can be attributed to the bacteria and EPS fibers attached to the electrode, resulting in higher interfacial resistance. The decreased Rct following the maximum coverage achieved by the biofilm on the electrode was most likely

Conclusions

A PPy enhanced flexible biofilm sensor has been successfully demonstrated and used to monitor the maturing development of P. aeruginosa biofilms by determining the Rct and the n of the adsorption CPE impedance associated with the biofilm. The Rct increases with time during the biofilms’ early stages of maturation and decreases during later stages of maturation. The n of the adsorption CPE impedance keeps increasing during early biofilm maturation and reaches a plateau afterward. The peak Rct

Acknowledgements

This work is supported by the grant of Flexible Electronics for Biological and Life Science Applications (FlexEBio) of the Integrative Graduate Education and Research Traineeship (IGERT) program of National Science Foundation under Grant No. DGE-0654112. The lithographic mask was fabricated with the help of Michael Skvarla at the Cornell NanoScale Facility, a member of the National Nanotechnology Infrastructure Network, which is supported by the National Science Foundation (Grant ECS-0335765).

Leo Y. Zheng received his BS and MS in Mechanical Engineering from Binghamton University in 2006 and 2008. Currently, Zheng is pursuing his PhD degree at Binghamton University as a member of Dr. Sammakia's research group. His research interest includes CFD in multiphase flows and thermal management, and flexible electronics for biological and medical applications. He was also a NSF IGRERT FlexEbio fellow from 2009 to 2012.

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Leo Y. Zheng received his BS and MS in Mechanical Engineering from Binghamton University in 2006 and 2008. Currently, Zheng is pursuing his PhD degree at Binghamton University as a member of Dr. Sammakia's research group. His research interest includes CFD in multiphase flows and thermal management, and flexible electronics for biological and medical applications. He was also a NSF IGRERT FlexEbio fellow from 2009 to 2012.

Robert B. Congdon received his BS and MA in biochemistry the University of Scranton in Scranton, PA in 2005 and 2007. He is a PhD candidate in Analytical Chemistry at Binghamton University. As a member of Prof. O.A. Sadik's research group, his research interests are in electroanalytical biosensors. Congdon is also a former NSF IGERT FlexEbio fellow from 2010 to 2012.

Omowunmi “Wunmi” Sadik is a Professor of Chemistry & Director, Center for Advanced Sensors & Environmental Systems at State University of New York at Binghamton. Dr. Sadik has held appointments at Harvard University, Cornell University and Naval Research Laboratories. Her research areas include interfacial molecular recognition processes, chemical & biosensors and their application to solving problems in biological system, energy and the environment. Dr. Sadik has authored over 130 research papers and 9 patents/patent applications. She has given 120 keynote and plenary lectures, and several invited lectures at national and international conferences and has contributed 150 conference lectures and posters.

Cláudia N. H. Marques is an Assistant Professor in the Department of Biological Sciences at Binghamton University. She has a MS in Medical Microbiology from the London School of Hygiene and Tropical Medicine, UK and, a PhD from the University of the West of England, UK. Her Research interests are single and multispecies biofilms, persister cell sub-populations, tolerance to antimicrobials and cell metabolic status. She recently was part of the team that isolated a fatty acid signaling molecule found to induce dispersion of biofilms. Currently her research focuses on the reversion of persister cells from a dormant to a metabolically active state.

David G. Davies is an associate professor in the Department of Biological Sciences at Binghamton University. He received his MS and PhD degrees in microbiology from the Center for Biofilm Engineering, Montana State University. Dr. Davies specializes in research on the physiology of biofilm bacteria.

Dr. Bahgat Sammakia is the vice president for research at Binghamton University and director of Small Scale Systems Integration and Packaging Center (S3IP), a New York State Center of Excellence. He is also a Distinguished SUNY professor of mechanical engineering in the Thomas J. Watson School of Engineering and Applied Science. Dr. Sammakia has over 200 published papers in refereed journals and conference proceedings, has contributed to five books in the areas of heat transfer and electronics packaging. Dr. Sammakia is an ASME fellow and a member of the IEEE. He is the editor of the Journal of Electronic Packaging, Transactions of the ASME.

Leann M. Lesperance, MD, PhD is Adjunct Clinical Associate Professor in the Decker School of Nursing, graduate program faculty in the Department of Bioengineering, Medical Director of the Clinical Science and Engineering Research Center, and a physician at the Decker Student Health Services Center, at Binghamton University. Her research involves biomedical devices and innovative technologies addressing chronic health problems.

James N. Turner received a BS in Engineering Science and a PhD in Biophysical Science from the State University of New York (SUNY) at Buffalo. He was a principle investigator at the Wadsworth Center, New York State's Department of Health and Prof. of Biomedical Sciences SUNY Albany and Adjunct Professor of Biomedical Engineering Renessalear Polytechnic Institute. He is presently a staff Research Scientist at Binghamton University. He has 130 full-length multidisciplinary publications applying the principles of engineering and physical sciences to biological and biomedical studies.

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