Real-time Monitoring of Bacterial Biofilms Metabolic Activity by a Redox-Reactive Nanosensors Array

Abstract Background Bacterial 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, separated by open water channels that act as a circulatory system that enable better diffusion of nutrients and easier removal of metabolic waste products. The monitoring of biofilms can provide important information on fundamental biofilm-related processes. That information can shed light on the bacterial processes and enable scientists to find ways of preventing future bacterial infections. Various approaches in use for biofilm analysis are based on microscopic, spectrochemical, electrochemical, and piezoelectrical methods. All 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. Results Here, detection and monitoring of the metabolic activity of bacterial biofilms in high-ionic-strength solutions were enabled as a result of novel surface modification by an active redox system, composed of 9,10-dihydroxyanthracene/9,10-anthraquinone, on the oxide layer of the SiNW, yielding a chemically-gated FET array. With the use of enzymatic reactions of oxidases, metabolites can be converted to Common.EditSubmissionSteps.Transform.EquationText and monitored by the nanosensors. Here, the successful detection of glucose metabolites in high-ionic-strength solutions, such as bacterial media, without pre-processing of small volume samples under different conditions and treatments, has been demonstrated. The biofilms were treated with antibiotics differing in their mechanisms of action and were compared to untreated biofilms. Further examination of biofilms under antibiotic treatment with SiNW-FET devices could shed light on the bioprocess that occurs within the biofilm. Moreover, finding proper treatment that eliminates the biofilm could be examined by the novel nanosensor as a monitoring tool. Conclusions To summarize, the combination of redox-reactive SiNW-FET devices with micro-fluidic techniques enables the performance of rapid, automated, and real-time metabolite detection with the use of minimal sample size, noninvasively and label-free. This novel platform can be used as an extremely sensitive tool for detection and establishing medical solutions for bacterial-biofilm eradication and for finding a proper treatment to eliminate biofilm contaminations. Moreover, the sensing system can be used as a research tool for further understanding of the metabolic processes that occur within the bacterial biofilm population.

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Real-time Monitoring of Bacterial Biofilms Metabolic Activity by a Redox-Reactive Nanosensors Array

10.21203/rs.3.rs-20969/v1 ◽

2020 ◽

Author(s):

Ella Yeor Davidi ◽

Marina Zverzhinetsky ◽

Vadim Krivitsky ◽

Fernando Patolsky

Keyword(s):

Ionic Strength ◽

Real Time ◽

Metabolic Activity ◽

High Ionic Strength ◽

Bacterial Biofilm ◽

Bacterial Biofilms ◽

Proper Treatment ◽

Real Time Monitoring ◽

Waste Products ◽

Shed Light

Abstract Background: Bacterial 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, separated by open water channels that act as a circulatory system that enable better diffusion of nutrients and easier removal of metabolic waste products. The monitoring of biofilms can provide important information on fundamental biofilm-related processes. That information can shed light on the bacterial processes and enable scientists to find ways of preventing future bacterial infections. Various approaches in use for biofilm analysis are based on microscopic, spectrochemical, electrochemical and piezoelectrical methods. All 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.Results: Here, detection and monitoring of the metabolic activity of bacterial biofilms in high-ionic-strength solutions were enabled as a result of novel surface modification by an active redox system, composed of 9,10-dihydroxyanthracene/9,10-anthraquinone, on the oxide layer of the SiNW, yielding a chemically-gated FET array. With the use of enzymatic reactions of oxidases, metabolites can be converted to and monitored by the nanosensors. Here, the successful detection of glucose consumption in high-ionic-strength solutions, such as bacterial media, without pre-processing of small volume samples under different conditions and treatments, has been demonstrated. The biofilms were treated with antibiotics differing in their mechanisms of action and were compared to untreated biofilms. Further examination of biofilms under antibiotic treatment with SiNW-FET devices could shed light on the bioprocess that occurs within the biofilm. Moreover, finding a proper treatment that eliminates the biofilm could be examined by the novel nanosensor array as a monitoring tool.Conclusions: To summarize, the combination of redox-reactive SiNW-FET devices with microfluidic techniques enables the performance of label-free, rapid, automated, multiplex and real-time noninvasive metabolites detection for the investigation of bacterial biofilms. This novel platform can be used as an extremely sensitive tool for detection and establishing medical solutions for bacterial-biofilm eradication and for finding a proper treatment to eliminate biofilm contaminations. Moreover, the sensing system can be used as a research tool for further understanding of the metabolic processes that occur within the bacterial biofilm population.

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Real-time monitoring of bacterial biofilms metabolic activity by a redox-reactive nanosensors array

Journal of Nanobiotechnology ◽

10.1186/s12951-020-00637-y ◽

2020 ◽

Vol 18 (1) ◽

Author(s):

Ella Yeor-Davidi ◽

Marina Zverzhinetsky ◽

Vadim Krivitsky ◽

Fernando Patolsky

Keyword(s):

Real Time ◽

Metabolic Activity ◽

Bacterial Biofilms ◽

Real Time Monitoring

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Specific detection of biomolecules in physiological solutions using graphene transistor biosensors

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1625010114 ◽

2016 ◽

Vol 113 (51) ◽

pp. 14633-14638 ◽

Cited By ~ 95

Author(s):

Ning Gao ◽

Teng Gao ◽

Xiao Yang ◽

Xiaochuan Dai ◽

Wei Zhou ◽

...

Keyword(s):

Ionic Strength ◽

Real Time ◽

Protein Detection ◽

High Ionic Strength ◽

Polymer Layer ◽

General Strategy ◽

Detection Of Biomolecules ◽

Permeable Polymer ◽

Graphene Devices ◽

Modified Graphene

Nanomaterial-based field-effect transistor (FET) sensors are capable of label-free real-time chemical and biological detection with high sensitivity and spatial resolution, although direct measurements in high–ionic-strength physiological solutions remain challenging due to the Debye screening effect. Recently, we demonstrated a general strategy to overcome this challenge by incorporating a biomolecule-permeable polymer layer on the surface of silicon nanowire FET sensors. The permeable polymer layer can increase the effective screening length immediately adjacent to the device surface and thereby enable real-time detection of biomolecules in high–ionic-strength solutions. Here, we describe studies demonstrating both the generality of this concept and application to specific protein detection using graphene FET sensors. Concentration-dependent measurements made with polyethylene glycol (PEG)-modified graphene devices exhibited real-time reversible detection of prostate specific antigen (PSA) from 1 to 1,000 nM in 100 mM phosphate buffer. In addition, comodification of graphene devices with PEG and DNA aptamers yielded specific irreversible binding and detection of PSA in pH 7.4 1x PBS solutions, whereas control experiments with proteins that do not bind to the aptamer showed smaller reversible signals. In addition, the active aptamer receptor of the modified graphene devices could be regenerated to yield multiuse selective PSA sensing under physiological conditions. The current work presents an important concept toward the application of nanomaterial-based FET sensors for biochemical sensing in physiological environments and thus could lead to powerful tools for basic research and healthcare.

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Real-time monitoring of cellular metabolic activity: intracellular oxygen

Nature Methods ◽

10.1038/nmeth.f.396 ◽

2016 ◽

Vol 13 (10) ◽

pp. i-ii

Author(s):

Andrea Krumm ◽

Conn Carey

Keyword(s):

Real Time ◽

Metabolic Activity ◽

Real Time Monitoring ◽

Cellular Metabolic Activity

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Evidence against a Simple Tethering Model for Enhancement of Herpes Simplex Virus DNA Polymerase Processivity by Accessory Protein UL42

Journal of Virology ◽

10.1128/jvi.76.20.10270-10281.2002 ◽

2002 ◽

Vol 76 (20) ◽

pp. 10270-10281 ◽

Cited By ~ 19

Author(s):

Murari Chaudhuri ◽

Deborah S. Parris

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Ionic Strength ◽

Real Time ◽

Dna Polymerase ◽

Dissociation Rate ◽

High Ionic Strength ◽

Apparent Affinity ◽

Simplex Virus ◽

Low Ionic Strength

ABSTRACT The DNA polymerase holoenzyme of herpes simplex virus type 1 (HSV-1) is a stable heterodimer consisting of a catalytic subunit (Pol) and a processivity factor (UL42). HSV-1 UL42 differs from most DNA polymerase processivity factors in possessing an inherent ability to bind to double-stranded DNA. It has been proposed that UL42 increases the processivity of Pol by directly tethering it to the primer and template (P/T). To test this hypothesis, we took advantage of the different sensitivities of Pol and Pol/UL42 activities to ionic strength. Although the activity of Pol is inhibited by salt concentrations in excess of 50 mM KCl, the activity of the holoenzyme is relatively refractory to changes in ionic strength from 50 to 125 mM KCl. We used nitrocellulose filter-binding assays and real-time biosensor technology to measure binding affinities and dissociation rate constants of the individual subunits and holoenzyme for a short model P/T as a function of the ionic strength of the buffer. We found that as observed for activity, the binding affinity and dissociation rate constant of the Pol/UL42 holoenzyme for P/T were not altered substantially in high- versus low-ionic-strength buffer. In 50 mM KCl, the apparent affinity with which UL42 bound the P/T did not differ by more than twofold compared to that observed for Pol or Pol/UL42 in the same low-ionic-strength buffer. However, increasing the ionic strength dramatically decreased the affinity of UL42 for P/T, such that it was reduced more than 3 orders of magnitude from that of Pol/UL42 in 125 mM KCl. Real-time binding kinetics revealed that much of the reduced affinity could be attributable to an extremely rapid dissociation of UL42 from the P/T in high-ionic-strength buffer. The resistance of the activity, binding affinity, and stability of the holoenzyme for the model P/T to increases in ionic strength, despite the low apparent affinity and poor stability with which UL42 binds the model P/T in high concentrations of salt, suggests that UL42 does not simply tether the Pol to DNA. Instead, it is likely that conformational alterations induced by interaction of UL42 with Pol allow for high-affinity and high-stability binding of the holoenzyme to the P/T even under high-ionic-strength conditions.

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Real-time monitoring of cadmium toxicity in rabbit kidney cells

Acta Veterinaria Brno ◽

10.2754/avb201584040351 ◽

2015 ◽

Vol 84 (4) ◽

pp. 351-356 ◽

Cited By ~ 3

Author(s):

Michal Milek ◽

Dana Marcinčáková ◽

Tomáš Csank ◽

Petra Kšonžeková ◽

Marcel Falis ◽

...

Keyword(s):

Toxic Effect ◽

Real Time ◽

Metabolic Activity ◽

Mtt Assay ◽

Metal Salt ◽

Cadmium Toxicity ◽

Rabbit Kidney ◽

Kidney Cells ◽

Dependent Manner ◽

Real Time Monitoring

The aim of this study was to investigate the toxic effect of the metal salt cadmium chloride dihydrate on the rabbit kidney cell line using the xCELLigence system or real-time cell analyser (RTCA), and to compare this relatively new method with standard biological cytotoxicity assays. This system provides real-time monitoring of cell behaviour and proliferative activity during the whole time of experiment. Moreover, after 24 h exposure of cells to cadmium, colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-difenyl tetrazolium bromide (MTT) test was used to measure the metabolic activity and cytotoxicity was determined by measurement of lactate dehydrogenase (LDH) leaked from damaged cells. We found that renal cells exposed to lower concentrations (5–10 mg·l-1) of cadmium tend to grow similarly to control cells, however, cell index was significantly different (P < 0.05) after 24 h. With increasing concentration of cadmium (15–50 mg·l-1) significantly lower proliferative (P < 0.05) and metabolic activity (P < 0.05) of cells was observed and cytotoxicity increased simultaneously (P < 0.001). In addition, we found that the real-time monitoring of the cell response was significantly correlated with commonly used biological methods for toxicity measurement, for MTT assay R2 was 0.9448 (P < 0.01) and for LDH assay R2 was 0.9466 (P < 0.01), respectively. The present study is the first report when combination of RTCA, MTT assay and LDH test was used for cadmium nephrotoxicity assessment. In all these methods, the toxic effect of cadmium on rabbit kidney cells increased in a concentration-dependent manner.

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