Redox-mediated Biomolecular information transfer in single electrogenetic biological cells.

Electronic communication in natural systems makes use, inter alia, of molecular transmission, where electron transfer occurs within networks of redox reactions, which play a vital role in many physiological systems. In view of the limited understanding of redox signaling, we developed an approach and an electrochemical-optical lab-on-a-chip to observe cellular responses in localized redox environments. The developed fluidic micro-system uses electrogenetic bacteria in which a cellular response is activated to electrically and chemically induced stimulations. Specifically, controlled environments for the cells are created by using microelectrodes to generate spatiotemporal redox gradients. The in-situ cellular responses at both single-cell and population levels are monitored by optical microscopy. The elicited electrogenetic fluorescence intensities after 210 min in response to electrochemical and chemical activation were 1.3 × 108±0.30 × 108 arbitrary units (A.U.) and 1.2 × 108±0.30 × 108 A.U. per cell population, respectively, and 1.05 ± 0.01 A.U. and 1.05 ± 0.01 A.U. per-cell, respectively. We demonstrated that redox molecules' mass transfer between the electrode and cells - and not the applied electrical field - activated the electrogenetic cells. Specifically, we found an oriented amplified electrogenetic response on the charged electrodes' downstream side, which was determined by the location of the stimulating electrodes and the flow profile. We then focused on the cellular responses and observed distinct subpopulations that were attributed to electrochemical rather than chemical stimulation, with the distance between the cells and the stimulating electrode being the main determinant. These observations provide a comprehensive understanding of the mechanisms by which diffusible redox mediators serve as electron shuttles, imposing context and activating electrogenetic responses.

On-Chip Electrochemical Sensing with an Enhanced Detecting Signal Due to Concentration Polarization-Based Analyte Preconcentration.

Here, we integrated two key technologies within a microfluidic system, an electrokinetic preconcentration of analytes by ion Concentration Polarization (CP) and local electrochemical sensors to detect the analytes, which can synergistically act to significantly enhance the detection signal. This synergistic combination, offering both decoupled and coupled operation modes for continuous monitoring, was validated by the intensified fluorescent intensities of CP-preconcentrated analytes and the associated enhanced electrochemical response using differential pulse voltammetry and chronoamperometry. The system performance was evaluated by varying the location of the active electrochemical sensor, target analyte concentrations, and electrolyte concentration using fluorescein molecules as the model analyte and Homovanillic acid (HVA) as the target bioanalyte within both phosphate-buffered saline (PBS) and artificial sweat solution. The combination of on-chip electrochemical sensing with CP-based preconcentration renders this generic approach adaptable to various analytes. This advanced system shows remarkable promise for enhancing biosensing detection in practical applications while bridging the gap between fundamental research and practical implementation.

Microfluidic channel sensory system for electro-addressing cell location, determining confluency, and quantifying a general number of cells.

Microfluidics is a highly useful platform for culturing, monitoring, and testing biological cells. The integration of electrodes into microfluidic channels extends the functionality, sensing, and testing capabilities of microfluidic systems. By employing an electrochemical impedance spectroscopy (EIS) technique, the non-invasive, label-free detection of the activities of cells in real-time can be achieved. To address the movement toward spatially resolving cells in cell culture, we developed a sensory system capable of electro-addressing cell location within a microfluidic channel. This simple system allows for real-time cell location, integrity monitoring (of barrier producing cells), and confluency sensing without the need for frequent optical evaluation-saving time. EIS results demonstrate that cells within microfluidic channels can be located between various pairs of electrodes at different positions along the length of the device. Impedance spectra clearly differentiates between empty, sparse, and confluent microfluidic channels. The system also senses the level of cell confluence between electrode pairs-allowing for the relative quantification of cells in different areas of the microfluidic channel. The system's electrode layout can easily be incorporated into other devices. Namely, organ-on-a-chip devices, that require the monitoring of precise cell location and confluency levels for understanding tissue function, modeling diseases, and for testing therapeutics.

Diffusion- and Chemometric-Based Separation of Complex Electrochemical Signals That Originated from Multiple Redox-Active Molecules.

In situ analysis of multiple biomarkers in the body provides better diagnosis and enables personalized health management. Since many of these biomarkers are redox-active, electrochemical sensors have shown promising analytical capabilities to measure multiple redox-active molecules. However, the analytical performance of electrochemical sensors rapidly decreases in the presence of multicomponent biofluids due to their limited ability to separate overlapping electrochemical signals generated by multiple molecules. Here we report a novel approach to use charged chitosan-modified electrodes to alter the diffusion of ascorbic acid, clozapine, L-homocysteine, and uric acid-test molecules with various molecular charges and molecular weights. Moreover, we present a complementary approach to use chemometrics to decipher the complex set of overlapping signals generated from a mixture of differentially charged redox molecules. The partial least square regression model predicted three out of four redox-active molecules with root mean square error, Pearson correlation coefficient, and R-squared values of 125 µM, 0.947, and 0.894; 51.8 µM, 0.877, and 0.753; 55.7 µM, 0.903, and 0.809, respectively. By further enhancing our understanding of the diffusion of redox-active molecules in chitosan, the in-situ separation of multiple molecules can be enabled, which will be used to establish guidelines for the effective separation of biomarkers.

Use of some cost-effective technologies for a routine clinical pathology laboratory.

Classically, the need for highly sophisticated instruments with important economic costs has been a major limiting factor for clinical pathology laboratories, especially in developing countries. With the aim of making clinical pathology more accessible, a wide variety of free or economical technologies have been developed worldwide in the last few years. 3D printing and Arduino approaches can provide up to 94% economical savings in hardware and instrumentation in comparison to commercial alternatives. The vast selection of point-of-care-tests (POCT) currently available also limits the need for specific instruments or personnel, as they can be used almost anywhere and by anyone. Lastly, there are dozens of free and libre digital tools available in health informatics. This review provides an overview of the state-of-the-art on cost-effective alternatives with applications in routine clinical pathology laboratories. In this context, a variety of technologies including 3D printing and Arduino, lateral flow assays, plasmonic biosensors, and microfluidics, as well as laboratory information systems, are discussed. This review aims to serve as an introduction to different technologies that can make clinical pathology more accessible and, therefore, contribute to achieve universal health coverage.

Electrochemical Determination of Hydroxyurea in a Complex Biological Matrix Using MoS2-Modified Electrodes and Chemometrics.

Hydroxyurea, an oral medication with important clinical benefits in the treatment of sickle cell anemia, can be accurately determined in plasma with a transition metal dichalcogenide-based electrochemical sensor. We used a two-dimensional molybdenum sulfide material (MoS2) selectively electrodeposited on a polycrystalline gold electrode via tailored waveform polarization in the gold electrical double layer formation region. The electro-activity of the modified electrode depends on the electrical waveform parameters used to electro-deposit MoS2. The concomitant oxidation of the MoS2 material during its electrodeposition allows for the tuning of the sensor's specificity. Chemometrics, utilizing mathematical procedures such as principal component analysis and multivariable partial least square regression, were used to process the electrochemical data generated at the bare and the modified electrodes, thus allowing the hydroxyurea concentrations to be predicted in human plasma. A limit-of-detection of 22 nM and a sensitivity of 37 nA cm-2 µM-1 were found to be suitable for pharmaceutical and clinical applications.

Probing antibody surface density and analyte antigen incubation time as dominant parameters influencing the antibody-antigen recognition events of a non-faradaic and diffusion-restricted electrochemical immunosensor.

Electrochemical sensors based on antibody-antigen recognition events are commonly used for the rapid, label-free, and sensitive detection of various analytes. However, various parameters at the bioelectronic interface, i.e., before and after the probe (such as an antibody) assembly onto the electrode, have a dominant influence on the underlying detection performance of analytes (such as an antigen). In this work, we thoroughly investigate the dependence of the bioelectronic interface characteristics on parameters that have not been investigated in depth: the antibody density on the electrode's surface and the antigen incubation time. For this important aim, we utilized the sensitive non-faradaic electrochemical impedance spectroscopy method. We showed that as the incubation time of the antigen-containing drop solution increased, a decrease was observed in both the solution resistance and the diffusional resistance with reflecting boundary elements, as well as the capacitive magnitude of a constant phase element, which decreased at a rate of 160 ± 30 kΩ/min, 800 ± 100 mΩ/min, and 520 ± 80 pF × s(α-1)/min, respectively. Using atomic force microscopy, we also showed that high antibody density led to thicker electrode coating than low antibody density, with root-mean-square roughness values of 2.2 ± 0.2 nm versus 1.28 ± 0.04 nm, respectively. Furthermore, we showed that as the antigen accumulated onto the electrode, the solution resistance increased for high antibody density and decreased for low antibody density. Finally, the antigen detection performance test yielded a better limit of detection for low antibody density than for high antibody density (0.26 µM vs 2.2 µM). Overall, we show here the importance of these two factors and how changing one parameter can drastically affect the desired outcome. Graphical abstract.

A platinum black-modified microelectrode for in situ olanzapine detection in microliter volumes of undiluted serum.

Olanzapine is a thienobenzodiazepine compound. It is one of the newer types of antipsychotic drugs used in the treatment of schizophrenia and other psychotic disorders. Several methods have been reported for analyzing olanzapine in its pure form or combined with other drugs and in biological fluids. These methods include high-performance liquid chromatography and liquid chromatography-tandem mass spectroscopy. Although many of the reported methods are accurate and sensitive, they require the use of sophisticated equipment, lack in situ analysis, and require expensive reagents. Moreover, several of these methods are cumbersome, require prolonged sample pretreatment, strict control of pH, and long reaction times. Here we present the development of a miniaturized electrochemical sensor that will enable minimally invasive, real-time, and in situ monitoring of olanzapine levels in microliter volumes of serum samples. For this purpose, we modified a microfabricated microelectrode with a platinum black film to increase the electrocatalytic activity of the microelectrode towards olanzapine oxidation; this improved the overall selectivity and sensitivity of the sensor. We observed in recorded voltammograms the anodic current dose response characteristics in microliter volumes of olanzapine-spiked serum samples that resulted in a limit of detection of 28.6 ± 1.3 nM and a sensitivity of 0.14 ± 0.02 µA/cm2 nM. Importantly, the platinum black-modified microelectrode exhibited a limit of detection that is below the clinical threshold (65-130 nM). Further miniaturizing and integrating such sensors into point-of-care devices provide real-time monitoring of olanzapine blood levels; this will enable treatment teams to receive feedback and administer adjustable olanzapine therapy.

A reduced-graphene oxide-modified microelectrode for a repeatable detection of antipsychotic clozapine using microliters-volumes of whole blood.

Antipsychotic clozapine is the most effective medication currently available for schizophrenia. However, clozapine is dramatically underutilized due to its harsh side effects that are not effectively monitored. By continuously monitoring clozapine blood levels, such as use of an implantable glucometer, which has transformed diabetes management, the treatment can be optimized and side effects will be minimized. Currently, none of the methods for clozapine detection show the ability to repeatedly measure clozapine in whole blood without pretreatment steps. Here we propose using a microelectrode modified with reduced graphene oxide-a material that was used for repeatable measurements in implantable electrochemical devices. We present the successful direct electrodeposition of reduced-graphene oxide coating onto microelectrodes. Systematic characterization of the electrodeposition technique parameters (i.e., the technique scan rate and the number of cycles) revealed their effect on the electrochemical activity and the structural properties (the film thickness and roughness) of the films. The developed reduced-graphene oxide-modified microelectrode exhibited the feasibility to detect clozapine in microliters-volume-samples of whole blood with a limit-of-detection and a sensitivity of 0.64 ±â€¯0.04 µM and 19.6 ±â€¯1.3 µA/cm2µM, respectively. Moreover, the reduced graphene oxide-modified microelectrodes exhibited high repeatability (retaining 94.6% of the electrochemical signal after 10 repeats), reproducibility (3.6% relative standard deviation), and storage stability (retaining 89% of the electrochemical signal after 4 weeks). Finally, relative recovery studies of 0.5, 1, and 2 µM clozapine concentrations resulted in 108 ±â€¯4.0%, 112 ±â€¯3.5%, and 103 ±â€¯2.2%, respectively. Future studies should investigate the microelectrode fouling mechanisms in whole blood and explore methods to overcome fouling.

Portable low-cost open-source wireless spectrophotometer for fast and reliable measurements.

We demonstrate a low-cost standalone portable spectrophotometer for fast and reliable measurement execution. The data acquired can be both displayed via a dedicated smartphone application or a computer interface, allowing users either to gather and view data on the move or set up a continuous experiment. All design and software files are open-source and are intended for the device to be easily replicable and further customizable to suit specific applications. The assembled device can measure absorption in the wavelength range from 450 nm to 750 nm with a resolution of 15 nm and is housed in a 90 × 85 × 58 mm casing. Validation of the device was carried out by assessing wavelength accuracy, dynamic range and the signal-to-noise ratio of the system, followed by testing in three different applications where limit of quantification, limit of detection and relative standard deviations were determined. The results indicated better performance than low-cost spectrophotometers, on average being comparable to moderate to high-cost spectrophotometers.

A Chitosan-Carbon Nanotube-Modified Microelectrode for In Situ Detection of Blood Levels of the Antipsychotic Clozapine in a Finger-Pricked Sample Volume.

The antipsychotic clozapine is the most effective medication available for schizophrenia and it is the only antipsychotic with a known efficacious clinical range. However, it is dramatically underutilized due to the inability to test clozapine blood levels in finger-pricked patients' samples. This prevents obtaining immediate blood levels information, resulting in suboptimal treatment. The development of an electrochemical microsensor is presented, which enables, for the first time, clozapine detection in microliters volume whole blood. The sensor is based on a microelectrode modified with micrometer-thick biopolymer chitosan encapsulating carbon nanotubes. The developed sensor detects clozapine oxidation current, in the presence of other electroactive species in the blood, which generate overlapping electrochemical signals. Clozapine detection, characterized in whole blood from healthy volunteers, displays a sensitivity of 32 ± 3.0 µA cm-2 µmol-1 L and a limit-of-detection of 0.5 ± 0.03 µmol L-1 . Finally, the developed sensor displays a reproducible electrochemical signal (0.6% relative standard deviation) and high storage stability (9.8% relative standard deviation after 8 days) in serum samples and high repeatability (9% relative standard deviation for the 5th repetition) in whole blood samples. By enabling the rapid and minimally invasive clozapine detection at the point-of-care, an optimal schizophrenia treatment is provided.

The effect of loading carbon nanotubes onto chitosan films on electrochemical dopamine sensing in the presence of biological interference.

In vivo monitoring of the neurotransmitter dopamine can potentially improve the diagnosis of neurological disorders and elucidate their underlying biochemical mechanisms. While electrochemical sensors can detect unlabeled dopamine molecules, their sensing performance is dramatically reduced by electrochemical currents generated by other, interfering molecules (e.g., uric acid) in the biological environment. To overcome this caveat, the surface of the sensor is often modified with electrocatalytic materials, which are encapsulated inside a polymeric film; however, the effect of the encapsulating film on the sensing performance of the electrode has not been systematically studied. This study characterizes the effect of loading carbon nanotubes (CNTs) onto a chitosan film on the electrochemical sensing performance of dopamine in the presence of uric acid. Higher CNT loading increases the diffusion and electron transfer rate coefficients of the sensor and, in the presence of uric acid, provides better sensitivity (3.00µALµmol-1 for 1.75% CNT loading, vs 0.01µALµmol-1 for 1% loading) but a poorer limit-of-detection (2.00µmolL-1vs 1.00, respectively), as reported here for the first time. These findings can help optimize the sensitivity and the limit-of-detection of electrochemical sensors in complex biofluids to enable an in vivo monitoring of dopamine and other redox-active molecules.

Blood Draw Barriers for Treatment with Clozapine and Development of a Point-of-Care Monitoring Device.

BACKGROUND: While clozapine (CLZ) is the most effective antipsychotic drug for schizophrenia treatment, it remains underused. In order to understand the barriers of frequent blood draws for white blood cell counts (WBCs) and clozapine levels, we developed a psychiatrist survey and began an integrative approach of designing a point-of-care device that could eventually have real-time monitoring with immediate results. METHODS: We ascertained barriers related to CLZ management and the acceptance of possible solutions by sending an anonymous survey to physicians in psychiatric practice (n=860). In parallel, we tested CLZ sensing using a prototype point-of-care monitoring device. RESULTS: 255 responses were included in the survey results. The two barriers receiving mean scores with the highest agreement as being a significant barrier were patient nonadherence to blood work and blood work's burden on the patient (out of 28). Among nine solutions, the ability to obtain lab results in the physician's office or pharmacy was top ranked (mean±sd Likert scale [4.0±1.0]). Physicians responded that a point-of-care device to measure blood levels and WBCs would improve care and increase CLZ use. Residents ranked point-of-care devices higher than older physicians (4.07±0.87 vs. 3.47±1.08, p<0.0001). Also, the prototype device was able to detect CLZ reliably in 1.6, 8.2, and 16.3 µg/mL buffered solutions. DISCUSSION: Survey results demonstrate physicians' desire for point-of-care monitoring technology, particularly among younger prescribers. Prototype sensor results identify that CLZ can be detected and integrated for future device development. Future development will also include integration of WBCs for a complete detection device.

The Binding Effect of Proteins on Medications and Its Impact on Electrochemical Sensing: Antipsychotic Clozapine as a Case Study.

Clozapine (CLZ), a dibenzodiazepine, is demonstrated as the optimal antipsychotic for patients suffering from treatment-resistant schizophrenia. Like many other drugs, understanding the concentration of CLZ in a patient's blood is critical for managing the patients' symptoms, side effects, and overall treatment efficacy. To that end, various electrochemical techniques have been adapted due to their capabilities in concentration-dependent sensing. An open question associated with electrochemical CLZ monitoring is whether drug-protein complexes (i.e., CLZ bound to native blood proteins, such as serum albumin (SA) or alpha-1 acid-glycoprotein (AAG)) contribute to electrochemical redox signals. Here, we investigate CLZ-sensing performance using fundamental electrochemical methods with respect to the impact of protein binding. Specifically, we test the activity of bound and free fractions of a mixture of CLZ and either bovine SA or human AAG. Results suggest that bound complexes do not significantly contribute to the electrochemical signal for mixtures of CLZ with AAG or SA. Moreover, the fraction of CLZ bound to protein is relatively constant at 31% (AAG) and 73% (SA) in isolation with varying concentrations of CLZ. Thus, electrochemical sensing can enable direct monitoring of only the unbound CLZ, previously only accessible via equilibrium dialysis. The methods utilized in this work offer potential as a blueprint in developing electrochemical sensors for application to other redox-active medications with high protein binding more generally. This demonstrates that electrochemical sensing can be a new tool in accessing information not easily available previously, useful toward optimizing treatment regimens.

The interplay of electrode- and bio-materials in a redox-cycling-based clozapine sensor.

We investigate gold, TiN, and platinum in combination with a chitosan-catechol-based redox-cycling system (RCS) for electrochemical detection of the antipsychotic clozapine. We have previously demonstrated the RCS for detection of clozapine in serum, but challenges remain regarding low signal-to-noise ratios. This can be mitigated by selection of electrode materials with beneficial surface morphologies and/or compositions. We employ cyclic voltammetry to assess the redox current generated by clozapine, and differentiate solely surface-area-based effects from clozapine-specific ones using a standard redox couple. We find that nano- and microstructured platinum greatly amplifies the clozapine signal compared to gold (up to 1490-fold for platinum black). However, the material performs poorly in the presence of chloride ions, and RCS modification provides no further amplification. The RCS combined with atomic-layer-deposited (ALD) TiN, on the other hand, increases the signal by 7.54 times, versus 2.86 times for RCS on gold, with a 9.2-fold lower variability, indicating that the homogenous and chemically inert properties of ALD-TiN may make it an ideal electrode material.

Molecular processes in an electrochemical clozapine sensor.

Selectivity presents a crucial challenge in direct electrochemical sensing. One example is schizophrenia treatment monitoring of the redox-active antipsychotic clozapine. To accurately assess efficacy, differentiation from its metabolite norclozapine-similar in structure and redox potential-is critical. Here, the authors leverage biomaterials integration to study, and effect changes in, diffusion and electron transfer kinetics of these compounds. Specifically, the authors employ a catechol-modified chitosan film, which the authors have previously presented as the first electrochemical detection mechanism capable of quantifying clozapine directly in clinical serum. A key finding in our present work is differing dynamics between clozapine and norclozapine once the authors interface the electrodes with chitosan-based biomaterial films. These additional dimensions of redox information can thus enable selective sensing of largely analogous small molecules.

Microfluidic Arrayed Lab-On-A-Chip for Electrochemical Capacitive Detection of DNA Hybridization Events.

A microfluidic electrochemical lab-on-a-chip (LOC) device for DNA hybridization detection has been developed. The device comprises a 3 × 3 array of microelectrodes integrated with a dual layer microfluidic valved manipulation system that provides controlled and automated capabilities for high throughput analysis of microliter volume samples. The surface of the microelectrodes is functionalized with single-stranded DNA (ssDNA) probes which enable specific detection of complementary ssDNA targets. These targets are detected by a capacitive technique which measures dielectric variation at the microelectrode-electrolyte interface due to DNA hybridization events. A quantitative analysis of the hybridization events is carried out based on a sensing modeling that includes detailed analysis of energy storage and dissipation components. By calculating these components during hybridization events the device is able to demonstrate specific and dose response sensing characteristics. The developed microfluidic LOC for DNA hybridization detection offers a technology for real-time and label-free assessment of genetic markers outside of laboratory settings, such as at the point-of-care or in-field environmental monitoring.

Fusing Sensor Paradigms to Acquire Chemical Information: An Integrative Role for Smart Biopolymeric Hydrogels.

The Information Age transformed our lives but it has had surprisingly little impact on the way chemical information (e.g., from our biological world) is acquired, analyzed and communicated. Sensor systems are poised to change this situation by providing rapid access to chemical information. This access will be enabled by technological advances from various fields: biology enables the synthesis, design and discovery of molecular recognition elements as well as the generation of cell-based signal processors; physics and chemistry are providing nano-components that facilitate the transmission and transduction of signals rich with chemical information; microfabrication is yielding sensors capable of receiving these signals through various modalities; and signal processing analysis enhances the extraction of chemical information. The authors contend that integral to the development of functional sensor systems will be materials that (i) enable the integrative and hierarchical assembly of various sensing components (for chemical recognition and signal transduction) and (ii) facilitate meaningful communication across modalities. It is suggested that stimuli-responsive self-assembling biopolymers can perform such integrative functions, and redox provides modality-spanning communication capabilities. Recent progress toward the development of electrochemical sensors to manage schizophrenia is used to illustrate the opportunities and challenges for enlisting sensors for chemical information processing.

Multidimensional mapping method using an arrayed sensing system for cross-reactivity screening.

When measuring chemical information in biological fluids, challenges of cross-reactivity arise, especially in sensing applications where no biological recognition elements exist. An understanding of the cross-reactions involved in these complex matrices is necessary to guide the design of appropriate sensing systems. This work presents a methodology for investigating cross-reactions in complex fluids. First, a systematic screening of matrix components is demonstrated in buffer-based solutions. Second, to account for the effect of the simultaneous presence of these species in complex samples, the responses of buffer-based simulated mixtures of these species were characterized using an arrayed sensing system. We demonstrate that the sensor array, consisting of electrochemical sensors with varying input parameters, generated differential responses that provide synergistic information of sample. By mapping the sensing array response onto multidimensional heat maps, characteristic signatures were compared across sensors in the array and across different matrices. Lastly, the arrayed sensing system was applied to complex biological samples to discern and match characteristic signatures between the simulated mixtures and the complex sample responses. As an example, this methodology was applied to screen interfering species relevant to the application of schizophrenia management. Specifically, blood serum measurement of antipsychotic clozapine and antioxidant species can provide useful information regarding therapeutic efficacy and psychiatric symptoms. This work proposes an investigational tool that can guide multi-analyte sensor design, chemometric modeling and biomarker discovery.

Effect of electrical energy on the efficacy of biofilm treatment using the bioelectric effect.

BACKGROUND/OBJECTIVES: The use of electric fields in combination with small doses of antibiotics for enhanced treatment of biofilms is termed the 'bioelectric effect' (BE). Different mechanisms of action for the AC and DC fields have been reported in the literature over the last two decades. In this work, we conduct the first study on the correlation between the electrical energy and the treatment efficacy of the bioelectric effect on Escherichia coli K-12 W3110 biofilms. METHODS: A thorough study was performed through the application of alternating (AC), direct (DC) and superimposed (SP) potentials of different amplitudes on mature E. coli biofilms. The electric fields were applied in combination with the antibiotic gentamicin (10 µg/ml) over a course of 24 h, after the biofilms had matured for 24 h. The biofilms were analysed using the crystal violet assay, the colony-forming unit method and fluorescence microscopy. RESULTS: Results show that there is no statistical difference in treatment efficacy between the DC-, AC- and SP-based BE treatment of equivalent energies (analysis of variance (ANOVA) P>0.05) for voltages <1 V. We also demonstrate that the efficacy of the BE treatment as measured by the crystal violet staining method and colony-forming unit assay is proportional to the electrical energy applied (ANOVA P<0.05). We further verify that the treatment efficacy varies linearly with the energy of the BE treatment (r2 =0.984). Our results thus suggest that the energy of the electrical signal is the primary factor in determining the efficacy of the BE treatment, at potentials less than the media electrolysis voltage. CONCLUSIONS: Our results demonstrate that the energy of the electrical signal, and not the type of electrical signal (AC or DC or SP), is the key to determine the efficacy of the BE treatment. We anticipate that this observation will pave the way for further understanding of the mechanism of action of the BE treatment method and may open new doors to the use of electric fields in the treatment of bacterial biofilms.