Microchip based Impedance Sensors for in situ Evaluation of evaluation of Bacterial Biofilm Formation (See2B).
State of the art:
Biofilms are complex, highly variable, surface-associated communities of microorganisms, embedded in a self-produced matrix. Within biofilms bacteria are up to 1000 times more tolerant to antibiotics, disinfectants and other stresses. Several factors contribute to this increased tolerance, such as protection by the biofilm matrix, slow growth and low metabolic activity of bacteria within biofilms, biofilm heterogeneity and activation of stress response within biofilms. As a consequence biofilms cause very persistent contaminations and infections. Of all infections, 80% are biofilm associated. In the U.S. alone, the yearly direct medical cost of implant/device associated biofilm infections is estimated to exceed $3 billion, while the total cost of biofilms in industry is at least an order of magnitude higher. These costs are related to ineffective use of expensive antimicrobials, surgical replacement of medical devices, reduced efficiency of industrial processes, and waste/replacement of industrial equipment.
The tolerance of a specific biofilm is highly dependent on its specific structure and the type of antimicrobial used. E.g. early stage biofilms are in general more sensitive than older biofilms; biofilms without cellulose in the matrix are more sensitive to sodium hypochlorite ; … . Rapid detection of biofilms and accurate monitoring of biofilm structure can thus not only notify that antimicrobial treatment is required but could also give indications on the most effective type of antimicrobial treatment and monitor the clearance process. Today, there is a wide variety of techniques that allow in depth in vitro studies of biofilms from gene expression to microscopic characterization.[7, 8] However, there is a high need for techniques that allow in situ and in vivo detection and characterization of biofilms, e.g. on implants, on catheters, in industrial tanks and piping systems, … . These in situ characterization techniques would open the doors to ‘à la carte’ treatment of biofilm contaminations –i.e. personalized medicine in the case of health care- and drastically reduce the costs and problems related to biofilms. Yet, these are not available yet.
Objectives – Innovation - Impact:
The aim of this project is to deliver proof-of-concept for the application of electrochemical impedance sensors (microchip based) as an in situ tool to evaluate the gravity of a biofilm contamination or infection and to predict an effective, tailored anti-biofilm treatment. Hereto the following basic research questions need to be answered:
i) Can impedance measurement be used to determine the structure, dynamics (i.e. change of structure in time) and heterogeneity (i.e. variation of structure in space) of biofilms?
ii) If so, can biofilm impedance characteristics be correlated to tolerance against certain classes of antimicrobials and as such give indications of the gravity of the biofilm contamination and anti-biofilm treatments that are still effective?
iii) Can biofilm clearance by antimicrobials be monitored through impedance measurement?
Because of the huge socio-economic problems related to biofilm contaminations/infections and the great demand for in situ characterization of biofilms, the economic and scientific impact of this project, if successful, will be high.
Electrochemical impedance sensors measure (changes in) the electrical impedance of (micro)electrodes under an alternating electric field. Impedance measurement & analysis can be used to obtain physicochemical information on a system and has gained widespread use in monitoring several (biological) processes, such as enzyme activity, bio-molecular recognition events and presence/growth of free-living bacterial cells. In this project we want to investigate the use of microchip based impedance sensors for the detection and characterization of biofilms and the prediction of an effective anti-biofilm treatment. The innovative and creative nature of this project lies in the following three elements:
(i) Maximal exploitation of biofilm impedance data for detection, characterization and clearance
Very recently it has been shown that biofilm growth influences the impedance between microelectrodes. This provokes the tempting question as to which extent impedance measurement can be used to gain structural, dynamic and spatial information of biofilms and as such predict resistance to antimicrobials. Because of their importance for the biofilm tolerance, the structural features of interest are (i) strength of adhesion of biofilms cells, (ii) extent of bacterial cell division within biofilms and (iii) chemical and physical structure of the biofilm matrix. To analyze the effect of these features on the impedance, we will make use of genetic methods and simultaneous monitoring with more established techniques such as SEM, AFM, CLSM, and different staining methods. Biofilm tolerance is dependent on the biofilm structure.[2, 5, 10] If impedance measurement succeeds to yield information on the biofilm structure, a correlation between impedance characteristics and biofilm tolerance is expected. To elucidate a possible correlation, different classes of antimicrobials will be systematically tested against biofilms with varying impedance characteristics. Oppositely, the effect of antimicrobial treatment on the impedance characteristics will be studied to assess the potential of impedance measurement in monitoring biofilm clearance.
(ii) Increased resolution through miniaturization – microelectrodes on microchips
Our collaborators at Imec are developing a next generation impedance sensor using CMOS technology and silicon processing enabling an increased level of miniaturization. The increased miniaturization will likely provide an ideal spatial resolution for studying biofilm structure and heterogeneity on an unprecedented level.
(iii) Integrated microsystems for the highest applicability
Because of the strong integration capabilities of silicon IC devices, several technologies can be integrated into small microsystems, which can be developed for virtually every application (biofilm detection on implants, piping systems, …).
In order to deliver proof-of-concept for the application of electrochemical impedance sensors as an in situ tool to evaluate the gravity of a biofilm contamination and to predict an effective anti-biofilm treatment we will answer the following questions, each in a separate work package (WP).
-Work package 1. Can impedance measurement be used to determine the structure, dynamics (i.e. change of structure in time) and heterogeneity (i.e. variation of structure in space) of biofilms?
WP1.1: Global exploration of variation of impedance in time and space during biofilm formation
Biofilm formation is a complex dynamic process (called the biofilm life cycle) starting with the reversible attachment of a number of individual bacteria to a surface, followed by a stronger, irreversible adhesion of these bacteria, cell division and microcolony formation, matrix production and development into a complex 3D structure and dispersion of cells from the biofilm. As several events in the biofilm cycle could possibly affect the impedance characteristics, we prefer to follow the impedance during the whole course of this process. Salmonella Typhimurium is the preferred model organism, since the CMPG has extensive expertise in the genetics, visualization and eradication of its biofilm formation (e.g. [10, 12-14]). Preliminary experiments already indicated that Salmonella can dynamically form biofilms on different types of impedance sensor surfaces produced by Imec.
To explore the effect of biofilm formation on impedance, we will measure and analyze the effect of the different events of the Salmonella biofilm cycle on the impedance and the different components of impedance in function of the electrical current frequency applied between microelectrode and the reference electrode. To be able to correlate the biofilm events with changes in impedance, we will simultaneously follow biofilm formation by CLSM. An equivalent circuit model will be built to interpret the measured impedance data in terms of the electrical properties (capacitance, resistance) of the different subparts of the biofilm system. This will give us on the one hand an electrical fingerprint of the different components of the biofilm and on the other hand will generate more insight in the heterogeneous built-up of the biofilm. Different types of electrode materials, electrode distribution patterns, electrode topologies and magnitudes of electrode polarization will be evaluated in order to determine which types of sensors are best fitted to study certain biofilm cycle events and biofilm characteristics, including biofilm heterogeneity. The fact that Imec has experience in studying the attachment and electrical readout of eukaryotic cells (stem cells, cardiac cells and neurons) to electrodes promotes the feasibility of this study. However, Imec has never studied the effect of dynamic biological processes and structural features of biofilms on impedance, highlighting the challenge and creativity of this project.
WP1.2 Contribution of specific structural biofilm features to impedance characteristics
After having established a general picture of the effect of different life cycle events on impedance (and its components) in function of the different parameters (current frequency, electrode material, size, distribution, … ), we will refine our view and models on the effect of specific structural features:
-To determine the influence of the bacterial adhesion force on impedance (especially in the early phases of biofilm formation), we will compare impedance measurements to AFM measurements (in collaboration with prof. Y. Dufrêne (UCL)) of bacterial adhesion forces.
-The effect of cell division on impedance will be assessed by relating impedance data to CLSM time laps experiments. To easily follow cell division microscopically, a set of Salmonella cells with different combinations of genomic fluorescent labels will be used. Arrest of cell division can in the early biofilm stages probably be induced by chloramphenicol treatment.
-The effect of (chemistry and physics) of several matrix components (such as cellulose, fimbriae, flagella, eDNA, …) will be studied by measuring the biofilm impedance of knock-out mutants and overexpression constructs for different types of matrix components. Many of these genetic constructs are present at CMPG; expertise for construction of additional constructs is available. These experiments will be complemented by staining techniques for matrix components and SEM measurements.
-WP2. Can biofilm impedance characteristics predict tolerance/susceptibility against certain classes of antimicrobials?
To elucidate a possible correlation, several classes of antimicrobials (disinfectants and antibiotics with different structures and mode of action mechanisms) will be systematically tested against biofilms with varying impedance characteristics.
-WP3: Can biofilm clearance by antimicrobials be monitored through impedance measurement?
The effect of antimicrobial treatment on the impedance characteristics will be studied to assess the potential of impedance measurement in monitoring biofilm clearance. Hereto the impedance of treated biofilms will be compared with complementary data on biofilm clearance (CLSM, live dead staining, colony plate counting, …).