Impedimetric Sensors for Bacteria Detection

The application of electrochemical biosensors based on impedance detection has grown during the past years due to their high sensitivity and rapid response, making this technique extremely useful to detect biological interactions with biosensor platforms. This chapter is focused on the use of electrochemical impedance spectroscopy (EIS) for bacterial detection in two ways. On one hand, bacteria presence may be determined by the detection of metabolites produced by bacterial growth involving the media conductivity changes. On the other hand, faster and more selective bacterial detection may be achieved by the immobilization of bacteria on a sensor surface using biorecognition elements (antibodies, antimicrobial peptides, aptamers, etc.) and registering changes produced in the charge transfer resistance (faradic process) or interfacial impedance (nonfaradic process). Here we discuss different types of impedimetric biosensors for microbiological applications, making stress on their most important parameters, such as detection limits, detection times, selectivity, and sensitivity. The aim of the paper was to give a critical review of recent publications in the field and mark the future trends

Impedimetric antimicrobial peptide-based sensor for the early detection of periodontopathogenic bacteria

Peri-implantitis, an inflammation caused by biofilm formation, constitutes a major cause of implant failure in dentistry. Thus, the detection of bacteria at the early steps of biofilm growth represents a powerful strategy to prevent implant-related infections. In this regard, antimicrobial peptides (AMPs) can be used as effective biological recognition elements to selectively detect the presence of bacteria. Thus, the aim of the present study was to combine the use of miniaturized and integrated impedimetric transducers and AMPs to obtain biosensors with high sensitivity to monitor bacterial colonization. Streptococcus sanguinis, which is one of the most prevalent strains in the onset of periodontal diseases, was used as a model of oral bacteria. To this end, a potent AMP derived from human lactoferrin was synthesized and covalently immobilized on interdigitated electrode arrays (IDEA). X-ray photoelectron spectroscopy (XPS) and electrochemical impedance spectroscopy (EIS) were employed to optimize and characterize the method of immobilization. Noteworthy, the interaction of Streptococcus sanguinis with AMP-coated sensors provoked significant changes in the impedance spectra, which were univocally associated with the presence of bacteria, proving the feasibility of our method. In this regard, the developed biosensor permits to detect the presence of bacteria at concentrations starting from 101 colony forming units (CFU) mL-1 in KCl and from 102 CFU mL-1 in artificial saliva. Moreover, the system was devoid of cytotoxicity for human fibroblasts. These results indicate that the proposed approach can be effective in the detection of initial stages of biofilm formation, and may be useful in the early prevention and treatment of peri-implantitisPeer ReviewedPostprint (author's final draft

Interdigitated electrode arrays (idea) impedimetric transducers for bacterial biosensing applications

La tecnologia dels biosensors, basada en dispositius analítics que combinen un bioreceptor amb una unitat de transducció, s’ha aplicat en nombroses àrees de recerca per a la detecció de diferents analits d’interès. Els bacteris, especialment els bacteris patògens, són agents biològics importants per ser detectats en diversos camps com el diagnòstic clínic, la indústria alimentària o la qualitat de l’aigua per prevenir malalties en els éssers humans. No obstant això, els bacteris també es poden utilitzar en un ampli ventall d’aplicacions; per exemple, com a indicadors biològics per determinar la toxicitat de diversos compostos. En aquesta tesi es proposa l’ús de transductors impedimètrics basats en elèctrodes de tipus interdigitat (interdigitated electrode arrays, IDEA de les sigles en anglès) com a instrument per al desenvolupament d’aplicacions biosensores bacterianes. L’espectroscòpia electroquímica d’impedància és una tècnica àmpliament estudiada per caracteritzar biosistemes perquè permet monitoritzar els fenòmens que tenen lloc a la superfície dels elèctrodes. Aquesta tècnica no requereix marcadors en el procés de transducció i pot ser usada en un mode d’operació sense marcatge addicional; així se simplifiquen els assajos de biomonitoratge. Entre els diferents tipus de transductors impedimètrics, els elèctrodes de tipus interdigitat són realment avantatjosos en termes de miniaturització, d’obtenció d’una resposta ràpida i estable o d’increment en la relació senyal-soroll. La utilització dels dispositius IDEA com a base de transducció per a un biosensor permet reduir el temps i cost per assaig. A més a més, en aquest treball es detalla i demostra l’aplicabilitat dels IDEA tridimensionals (3D-IDEA), en els quals els dígits dels elèctrodes estan separats per barreres aïllants, que permeten millorar la sensibilitat en el registre de canvis superficials si els comparem amb els IDEA convencionals per a la detecció de bactèries. Els objectius d’aquest treball són l’elaboració i la validació d’estratègies de biodetecció, estables i reproduïbles, utilitzant IDEA i 3D-IDEA per a la identificació de bacteris com a analit d’interès o bé com a element de sensat. En el primer cas, s’ha dut a terme la detecció de bacteris o endotoxines bacterianes en mostres líquides, mentre que en el segon s’ha desenvolupat un biosensor del tipus microbià. Per tal de dur-ho a terme, els dispositius IDEA s’han (bio)funcionalitzat mitjançant diverses metodologies per desenvolupar en quatre aplicacions.Biosensor technology, consisting of analytical devices that conjugate a bioreceptor with a transducer unit, has been applied in numerous research areas for the detection of different analytes of interest. Bacteria, especially pathogenic bacteria, are important targets to be sensed and identified in many fields, like clinical diagnosis, food industry or water safety, to prevent a great number of diseases in humans. However, bacteria can be employed in a wide range of beneficial applications, such as their use as biological indicators to determine the toxicity of various compounds. In this thesis, the use of impedimetric transducers based on interdigitated electrode arrays (IDEA) has been proposed as a tool for the development of bacterial biosensing applications. Electrochemical Impedance Spectroscopy is a widely studied technique to characterize biosystems because it allows to monitor electrical events occurring on the surface of electrodes. This technique does not require additional markers for the transduction and can be used in a label-free operation mode and hence simplifying the biosensing assays. Among different types of impedimetric transducers interdigitated electrodes arrays are really advantageous in terms of easy-miniaturization, fast establishment of the steady-state signal response and increased signal-to-noise ratio. The utilization of IDEA devices as a base of a biosensor transducer permits reducing the time and cost per assay. In addition, the applicability of three-dimensional IDEA (3D-IDEA) is described and demonstrated, in which the electrode digits are separated by insulating barriers, to improve the sensitivity for the registration of superficial parameters compared with standard IDEA for bacteria sensing. The main aim of this work is the elaboration and testing of robust and reproducible biosensing strategies using IDEA and 3D-IDEA impedance transducers with bacteria, as an analyte target or as a sensing element. In the first case, the detection of bacteria or bacterial endotoxins in liquid samples may be performed and, in the second one, novel microbial-based biosensors may be developed. To this end, IDEA devices have been (bio)functionalized using various grafting schemes for their use in four different applications

Interdigitated electrode arrays (idea) impedimetric transducers for bacterial biosensing applications /

La tecnologia dels biosensors, basada en dispositius analítics que combinen un bioreceptor amb una unitat de transducció, s'ha aplicat en nombroses àrees de recerca per a la detecció de diferents analits d'interès. Els bacteris, especialment els bacteris patògens, són agents biològics importants per ser detectats en diversos camps com el diagnòstic clínic, la indústria alimentària o la qualitat de l'aigua per prevenir malalties en els éssers humans. No obstant això, els bacteris també es poden utilitzar en un ampli ventall d'aplicacions; per exemple, com a indicadors biològics per determinar la toxicitat de diversos compostos. En aquesta tesi es proposa l'ús de transductors impedimètrics basats en elèctrodes de tipus interdigitat (interdigitated electrode arrays, IDEA de les sigles en anglès) com a instrument per al desenvolupament d'aplicacions biosensores bacterianes. L'espectroscòpia electroquímica d'impedància és una tècnica àmpliament estudiada per caracteritzar biosistemes perquè permet monitoritzar els fenòmens que tenen lloc a la superfície dels elèctrodes. Aquesta tècnica no requereix marcadors en el procés de transducció i pot ser usada en un mode d'operació sense marcatge addicional; així se simplifiquen els assajos de biomonitoratge. Entre els diferents tipus de transductors impedimètrics, els elèctrodes de tipus interdigitat són realment avantatjosos en termes de miniaturització, d'obtenció d'una resposta ràpida i estable o d'increment en la relació senyal-soroll. La utilització dels dispositius IDEA com a base de transducció per a un biosensor permet reduir el temps i cost per assaig. A més a més, en aquest treball es detalla i demostra l'aplicabilitat dels IDEA tridimensionals (3D-IDEA), en els quals els dígits dels elèctrodes estan separats per barreres aïllants, que permeten millorar la sensibilitat en el registre de canvis superficials si els comparem amb els IDEA convencionals per a la detecció de bactèries. Els objectius d'aquest treball són l'elaboració i la validació d'estratègies de biodetecció, estables i reproduïbles, utilitzant IDEA i 3D-IDEA per a la identificació de bacteris com a analit d'interès o bé com a element de sensat. En el primer cas, s'ha dut a terme la detecció de bacteris o endotoxines bacterianes en mostres líquides, mentre que en el segon s'ha desenvolupat un biosensor del tipus microbià. Per tal de dur-ho a terme, els dispositius IDEA s'han (bio)funcionalitzat mitjançant diverses metodologies per desenvolupar en quatre aplicacions.Biosensor technology, consisting of analytical devices that conjugate a bioreceptor with a transducer unit, has been applied in numerous research areas for the detection of different analytes of interest. Bacteria, especially pathogenic bacteria, are important targets to be sensed and identified in many fields, like clinical diagnosis, food industry or water safety, to prevent a great number of diseases in humans. However, bacteria can be employed in a wide range of beneficial applications, such as their use as biological indicators to determine the toxicity of various compounds. In this thesis, the use of impedimetric transducers based on interdigitated electrode arrays (IDEA) has been proposed as a tool for the development of bacterial biosensing applications. Electrochemical Impedance Spectroscopy is a widely studied technique to characterize biosystems because it allows to monitor electrical events occurring on the surface of electrodes. This technique does not require additional markers for the transduction and can be used in a label-free operation mode and hence simplifying the biosensing assays. Among different types of impedimetric transducers interdigitated electrodes arrays are really advantageous in terms of easy-miniaturization, fast establishment of the steady-state signal response and increased signal-to-noise ratio. The utilization of IDEA devices as a base of a biosensor transducer permits reducing the time and cost per assay. In addition, the applicability of three-dimensional IDEA (3D-IDEA) is described and demonstrated, in which the electrode digits are separated by insulating barriers, to improve the sensitivity for the registration of superficial parameters compared with standard IDEA for bacteria sensing. The main aim of this work is the elaboration and testing of robust and reproducible biosensing strategies using IDEA and 3D-IDEA impedance transducers with bacteria, as an analyte target or as a sensing element. In the first case, the detection of bacteria or bacterial endotoxins in liquid samples may be performed and, in the second one, novel microbial-based biosensors may be developed. To this end, IDEA devices have been (bio)functionalized using various grafting schemes for their use in four different applications

Impedimetric label-free sensor for specific bacteria endotoxin detection by surface charge registration

An impedimetric sensor based on a three dimensional electrode array modified with concanavalin A (Con A) was used for label-free detection of bacterial endotoxin: lipopolysaccharide (LPS) from Escherichia coli. The transducer permits the detection of the surface charge changes due to interaction of immobilized Con A biorecognition element and LPS of E. coli in test solution. The deposition of Con A on the surface was carried out using the layer-by-layer method with polyethyleneimine (PEI) polycation as an initial layer. The sensor surface characterization by means of electrochemical impedance spectroscopy technique allowed registering variations in superficial resistance provoked by surface charge changes and is demonstrated as an effective method to monitor sensor parameters at each modification step as well as to follow Con A – LPS reaction. In order to prevent non-specific adsorption of LPS on PEI covered surface different blocking strategies were tested to achieve the specific response between Con A and LPS. Results obtained in this work clearly show that blocking with bovine serum albumin (BSA) is not sufficient to prevent non-specific interactions of PEI and to ensure the selective biorecognition of LPS by Con A. To achieve more efficient PEI blocking a new method was proposed based on consecutive deposition of Con A-glycogen-Con A layers. Sensors modified with PEI-(Con A-Gly)2-Con A multilayers are shown to be highly sensitive, selective and reproducible. Presented biosensor is able to detect bacterial LPS in a very short detection time (20 min) with 2 μg mL−1 limit of detection, which is much lower than reported for other biosensors with Con A.The authors acknowledge financial support from the Spanish Ministry of Economy and Competitiveness (project CTQ2014-54553-C3-1-R and S.B.O. fellowship of FPI-MICINN program BES-2015-071250) co-funded by the European Regional Development Fund (Feder) and from the Government of Russian Federation (Grant 074-U01).Peer reviewe

3D Impedimetric Biosensor for Cyanobacteria Detection in Natural Water Sources

The excessive growth of cyanobacteria in freshwater sources produces the development of toxic blooms mainly due to the production of cyanotoxins. Here, a novel impedimetric biosensor based on a three-dimensional interdigitated electrode array (3D-IDEA) for detection of cyanobacteria cells is reported. The 3D-IDEA sensor surface biofunctionalization was performed by means of the layer-by-layer method using polyethyleneimine (PEI) as the anchoring layer and concanavalin A (Con A) as the bioreceptor to lipopolysaccharides of cyanobacteria cells. The developed PEI-Con A 3D-IDEA sensors show a linear response (R2 = 0.992) of the impedance changes (RS) versus the logarithm of cyanobacteria concentrations in the range of 102–105 cells·mL−1 with the detection limit of 100 cells·mL−1. Moreover, to prevent the interference from components that may be present in real water samples and minimize a possible sample matrix effect, a filtration methodology to recover cyanobacterial cells was developed. The proposed methodology allows 91.2% bacteria recovery, permitting to obtain results similar to controlled assays. The developed system can be used in aquatic environments to detect cyanobacteria and consequently to prevent the formation of blooms and the production of cyanotoxins. Con A can bind to most polysaccharides and so react with other types of bacteria. However, currently, on the market, it is not possible to find specific biorecognition elements for cyanobacteria. Taking into consideration the specificity of samples to be analyzed (natural water resources), it is difficult to expect high concentration of other bacteria. In this sense, the developed methodology may be used as an alarm system to select samples for more thorough and precise laboratory analysis

3D Impedimetric Biosensor for Cyanobacteria Detection in Natural Water Sources

The excessive growth of cyanobacteria in freshwater sources produces the development of toxic blooms mainly due to the production of cyanotoxins. Here, a novel impedimetric biosensor based on a three-dimensional interdigitated electrode array (3D-IDEA) for detection of cyanobacteria cells is reported. The 3D-IDEA sensor surface biofunctionalization was performed by means of the layer-by-layer method using polyethyleneimine (PEI) as the anchoring layer and concanavalin A (Con A) as the bioreceptor to lipopolysaccharides of cyanobacteria cells. The developed PEI-Con A 3D-IDEA sensors show a linear response (R2 = 0.992) of the impedance changes (RS) versus the logarithm of cyanobacteria concentrations in the range of 102–105 cells·mL−1 with the detection limit of 100 cells·mL−1. Moreover, to prevent the interference from components that may be present in real water samples and minimize a possible sample matrix effect, a filtration methodology to recover cyanobacterial cells was developed. The proposed methodology allows 91.2% bacteria recovery, permitting to obtain results similar to controlled assays. The developed system can be used in aquatic environments to detect cyanobacteria and consequently to prevent the formation of blooms and the production of cyanotoxins. Con A can bind to most polysaccharides and so react with other types of bacteria. However, currently, on the market, it is not possible to find specific biorecognition elements for cyanobacteria. Taking into consideration the specificity of samples to be analyzed (natural water resources), it is difficult to expect high concentration of other bacteria. In this sense, the developed methodology may be used as an alarm system to select samples for more thorough and precise laboratory analysis

3D impedimetric sensors as a tool for monitoring bacterial response to antibiotics

The presence of antimicrobial contaminants like antibiotics in the environment is a major concern because they promote the emergence and the spread of multidrug resistant bacteria. Since the conventional systems for the determination of bacterial susceptibility to antibiotics rely on culturing methods that require long processing times, the implementation of novel strategies is highly required for fast and point-of-care applications. Here the development and characterization of a novel label-free biosensing platform based on a microbial biosensor approach to perform antibiotic detection bioassays in diluted solution is presented. The microbial biosensor is based on a three-dimensional interdigitated electrode array (3D-IDEA) impedimetric transducer with immobilized E. coli bacteria. In 3D-IDEA to increase the sensitivity to superficial impedance changes the electrode digits are separated by insulating barriers. A novel strategy is employed to selectively immobilize bacteria in the spaces over the electrode digits between the barriers, referred to here as trenches, in order to concentrate bacteria, improve the reproducibility of the E. coli immobilization and increase the sensitivity for monitoring bacterial response. For effective attachment of bacteria within the trenches an initial anchoring layer of a highly charged polycation, polyethyleneimine (PEI), was used. To facilitate immobilization of bacteria within the trenches and prevent their deposition on top of the barriers an important novelty is the use of poly(N-isopropylmethacrylamide) p(NIPMAM) microgels working as antifouling agents, deposited on top of the barriers by microcontact printing. The reported microbial biosensor approach allows the bacterial response to ampicillin, a bacteriolytic antibiotic, to be registered by means of impedance variations in a rapid and label-free operation that enables new possibilities in bioassays for toxicity testing.The authors acknowledge financial support from the Spanish Ministry of Economy and Competitiveness (project CTQ2014-54553-C3-1-R, CTQ2015-66254-C2-2-P and S. B. O fellowship BES-2015-071250). S. B. O. also acknowledges the PhD program in Biotechnology of Universitat Autònoma de Barcelona. O. M. is grateful for financial support from the Alexander von Humboldt FoundationWe acknowledge support of the publication fee by the CSIC Open Access Support Initiative through its Unit of Information Resources for Research (URICI

Novel impedimetric aptasensor for label-free detection of Escherichia coli O157:H7

Microbial safety of drinking water constitutes a major concern in countries at all levels of economic development. Thus, rapid, sensitive and cost effective methods of pathogenic bacteria detection, like common Escherichia coli O157:H7, which can cause important diseases, are highly required. In this work an impedimetric transducer modified with E. coli specific aptamer is studied. To enhance the sensitivity a three-dimensional interdigitated electrode array (3D-IDEA) impedimetric transducer, in which the electrodes are separated by insulating barriers was used. In this sensor chemical reactions at the surface of the barrier provoke electrical charge redistribution which causes changes in the surface conductivity. A DNA aptamer, which recognizes specifically the outer membrane proteins of the E. coli O157:H7, was selected as the biorecognition moiety. Here we report a novel label-free impedimetric aptasensor for detection and quantification of pathogenic E. coli O157:H7 with a low detection limit, good selectivity and short detection time. The developed sensor shows a linear response (R2 = 0.977), proportional to the logarithm of bacterial concentration present in the sample, with the limit of detection (LOD) of about 102 cfu mL−1. No response of the aptasensor was registered in the presence of other bacterial strains (E. coli k12, Salmonella typhimurium, Staphylococcus aureus), which confirms the selectivity of the suggested detection method. Additionally, the methodology of the aptasensor regeneration was developed, so that the detection may be performed several times with the same sensor. Moreover, suitability of the aptasensor for bacteria detection in real samples was demonstrated with a new approach involving bacteria pre-concentration.The authors acknowledge financial support from the Spanish Ministry of Economy and Competitiveness (project CTQ2014-54553-C3-1-R, CTQ2015-66254-C2-2-P and S.B.O fellowship of FPI-MICINN program BES-2015-071250) co-funded by the European Regional Development Fund (Feder) and from the Government of Russian Federation (Grant 074-U01). N.U. acknowledges funding from the People Programme (Marie Curie Actions) of the 7th Framework Programme of the European Union (FP7/2007-2013), TECNIOspring program from the Agency for Business Competitiveness of the Government of Catalonia (ACCIÓ).Peer reviewe

Impedimetric antimicrobial peptide-based sensor for the early detection of periodontopathogenic bacteria

Peri-implantitis, an inflammation caused by biofilm formation, constitutes a major cause of implant failure in dentistry. Thus, the detection of bacteria at the early steps of biofilm growth represents a powerful strategy to prevent implant-related infections. In this regard, antimicrobial peptides (AMPs) can be used as effective biological recognition elements to selectively detect the presence of bacteria. Thus, the aim of the present study was to combine the use of miniaturized and integrated impedimetric transducers and AMPs to obtain biosensors with high sensitivity to monitor bacterial colonization. Streptococcus sanguinis, which is one of the most prevalent strains in the onset of periodontal diseases, was used as a model of oral bacteria. To this end, a potent AMP derived from human lactoferrin was synthesized and covalently immobilized on interdigitated electrode arrays (IDEA). X-ray photoelectron spectroscopy (XPS) and electrochemical impedance spectroscopy (EIS) were employed to optimize and characterize the method of immobilization. Noteworthy, the interaction of Streptococcus sanguinis with AMP-coated sensors provoked significant changes in the impedance spectra, which were univocally associated with the presence of bacteria, proving the feasibility of our method. In this regard, the developed biosensor permits to detect the presence of bacteria at concentrations starting from 101 colony forming units (CFU) mL-1 in KCl and from 102 CFU mL-1 in artificial saliva. Moreover, the system was devoid of cytotoxicity for human fibroblasts. These results indicate that the proposed approach can be effective in the detection of initial stages of biofilm formation, and may be useful in the early prevention and treatment of peri-implantitisPeer Reviewe