Nanostructures, such as nanowires, with unique one-dimensional morphology, are very interesting materials due to their electron-transport properties. Nanowires with predictable and controllable conductance are very important for electrical and electronic applications and serve as critical building blocks for emerging nanotechnologies[1]. Their one-dimensionality leads to an extremely high surface-to-volume ratio, which makes them great field-effect-transistor (FET) devices. Changes in the electric field at their surfaces lead to depletion or accumulation of charge carriers at their "bulk", rendering them extremely sensitive to molecules adsorbed on their surfaces, to a point where single-molecule detection is made possible[2]. These nanostructures have led to the revolutionary concept of new devices for the detection of chemical and biological entities [3-27]. Moreover, they possess the ability to be ultra-sensitive, selective, and label-free real-time sensors.
Nonetheless, several detection limitations still need to be resolved in order to achieve the practical applicability of NW-based FET devices for sensing. One of the challenges related to sensing performance is the detection limitation due to Debye screening that occurs under conditions of high ionic strength. [18]. Under these conditions, such as physiological 155mM salt concentration, the screening length is about 1nm, masking charge alterations occurring on surface-bound receptor molecules (i.e. proteins or DNA linkers) that are kept away from the sensor surface by a distance of 2-12nm. Therefore, most reports regarding sensing with SiNW FETs have been carried out in solutions of low ionic strength. For optimal sensing, the Debye length must be carefully selected or samples must be diluted or desalted.
In developed countries, ninety percent of documented infections in hospitalized patients are caused by bacteria. The World Health Organization has estimated that each year, five million people die of bacterial infections, which are the second leading cause of death in the world (after cardiovascular diseases).
Bacteria often attach to surfaces and form dense aggregates called biofilms or bacterial mats. Biofilms are communities of surface-associated microorganisms living in cellular clusters or micro-colonies, encapsulated in a complex matrix composed of an extracellular polymeric substance (EPS), separated by open water channels that act as a circulatory system that enable better diffusion of nutrients and easier removal of metabolic waste products[28]. The National Institute of Health of the United States has estimated that more than 80% of the bacterial infections in the human population are biofilm-related, and that patient mortality associated with biofilms is substantial[29]. Under the protection of biofilm, microbial cells become tolerant and resistant to antibiotics and to the immune-system responses, which increases the difficulty of the clinical treatment of biofilm-based infections [30, 31]. Therefore, the effective treatment of biofilm infections with currently available antibiotics, while enabling evaluation of the treatment results, is a challenge that attracts the attention of the scientific community.
Techniques of real-time biofilm monitoring are based on a certain measured signal obtained from the biofilm under investigation. Signals such as acoustic waves, electrical fields, electric current, radiation (including light), or heat transfer can be investigated[32]. Monitoring techniques can be divided into direct measurements, related to the mass or the cell density, and indirect measurements of metabolic activity and products such as liquids or gases. Various approaches in use for biofilm analysis, from the beginning of formation to eradication, are based on microscopic, spectrochemical, electrochemical, and piezoelectrical methods[33-35]. These methods provide significant progress in understanding the bio-process related to biofilm formation and eradication. Nevertheless, the development of novel approaches for the real-time monitoring of biochemical, in particular metabolic activity, of bacterial species during the formation, life, and eradication of biofilms is of great potential importance.
haracterization of OMCs in a catalytically active microbial
biofilm. By measuring the electrochemical and spectroscopic
properties of microbial cells embedded in their natural
biofilm habitat, a more realistic picture on the natural
electron transfer will be provided. Therefore, we have
employed surface-enhanced resonance Raman (SERR) spec-
troscopy in combination with cyclic voltammetry (CV).
SERR spectroscopy exploits the combination of the molec-
ular resonance Raman (RR) and the surface-enhanced
Raman (SER) effect to probe selectively the heme groups
solely of the proteins in proximity of the electrode sur-
face.
[13, 14]
This powerful technique, in our case performed
under strict electrochemical control, reveals the redox,
coordination and spin states of the heme iron as well as the
nature of its axial ligand, thereby providing important
structural information that may complement the interpreta-
tion of electrochemical data obtained by CV
employed surface-enhanced resonance Raman (SERR) spec-
troscopy in combination with cyclic voltammetry (CV).
SERR spectroscopy exploits the combination of the molec-
ular resonance Raman (RR) and the surface-enhanced
Raman (SER) effect to probe selectively the heme groups
solely of the proteins in proximity of the electrode sur-
face.
[13, 14]
This powerful technique, in our case performed
under strict electrochemical control, reveals the redox,
coordination and spin states of the heme iron as well as the
nature of its axial ligand, thereby providing important
structural information that may complement the interpreta-
tion of electrochemical data obtained by CV
employed surface-enhanced resonance Raman (SERR) spec-
troscopy in combination with cyclic voltammetry (CV).
SERR spectroscopy exploits the combination of the molec-
ular resonance Raman (RR) and the surface-enhanced
Raman (SER) effect to probe selectively the heme groups
solely of the proteins in proximity of the electrode sur-
face.
[13, 14]
This powerful technique, in our case performed
under strict electrochemical control, reveals the redox,
coordination and spin states of the heme iron as well as the
nature of its axial ligand, thereby providing important
structural information that may complement the interpreta-
tion of electrochemical data obtained by CV
haracterization of OMCs in a catalytically active microbial
biofilm. By measuring the electrochemical and spectroscopic
properties of microbial cells embedded in their natural
biofilm habitat, a more realistic picture on the natural
electron transfer will be provided. Therefore, we have
employed surface-enhanced resonance Raman (SERR) spec-
troscopy in combination with cyclic voltammetry (CV).
SERR spectroscopy exploits the combination of the molec-
ular resonance Raman (RR) and the surface-enhanced
Raman (SER) effect to probe selectively the heme groups
solely of the proteins in proximity of the electrode sur-
face.
[13, 14]
This powerful technique, in our case performed
under strict electrochemical control, reveals the redox,
coordination and spin states of the heme iron as well as the
nature of its axial ligand, thereby providing important
structural information that may complement the interpreta-
tion of electrochemical data obtained by
Hence, biofilm monitoring by measurements of metabolic-activity products can supply information about the nature of bacterial biofilm. Strategies for non-invasive measurement of metabolic activity are diverse. For example, NMR was used decades ago to investigate substrate consumption in bacterial biofilms and to assess the effect of biofilm formation on the hydrodynamics of the surrounding liquid[36]. Bioluminescence and fluorescence methods demonstrated high sensitivity capabilities, but require labeled samples for monitoring[37-40]. The investigation and analysis of microbial metabolic activity through analysis of extracellular protein expression was demonstrated by protein-gel analysis. All the monitoring methods mentioned above are reliable and useful for biofilm research, but, on the other hand, every one of them still possesses disadvantages such as labeling, time-consuming, cost, and massive machines required for steps in the analysis, complex preparation of the detected sample or low sensitivity. [41, 42]. To overcome these limitations a new technology that combines real-time detection, label-free, selective and sensitive capabilities is still required.
Cellular metabolism, and in particular glucose metabolism, has been shown to reflect the state of living cells and microorganisms. More specifically, the inhibition of bacterial metabolism of glucose consumption can be used as an index of bacterial susceptibility to drugs [43, 44]. A unique redox-reactive modification on the surface of the SiNW FET devices was previously shown to be a powerful tool for glucose sensing in high-ionic-strength solution[13, 27]. The redox system’s functional group comprising 9,10-dihydroxyanthracene (AQ), that can be oxidized or reduced in a reversible manner, which involves a significant change in the charge[13, 45], and influences the conductivity of the FET. Hydrogen peroxide (H2O2) is an oxidative species that can selectively react with the redox modification and change the level of oxidation of the system. Moreover, hydrogen peroxide has been used in a technique for real-time monitoring of metabolites by the use of corresponding oxidase enzymes to convert target metabolites to H2O2[13]. This platform was coupled with microfluidic technologies that allowed the delivery of small volumes of detected samples on the surface of the nanosensor. Based on this work, we developed a platform that enables monitoring of the metabolic activity of biofilms based on glucose consumption, testing their susceptibility to drugs and other eradication efforts, to adjust the proper medical treatment to overcome biofilm infections. We believe that among the many different technologies available for monitoring biofilm growth, the SiNW-FET array is the most promising approach, as it affords direct, label-free, real-time, highly sensitive and specific monitoring of biofilm processes in a continuous, nondestructive manner, in their natural environment.