Name: manufacturing of a nafion-coated, reduced graphene oxide/polyaniline chemiresistive sensor to monitor ph in real-time during microbial fermentation
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Manufacturing of a Nafion-coated, Reduced Graphene Oxide/Polyaniline Chemiresistive Sensor to Monitor pH in Real-time During Microbial Fermentation at JoVE.co

The authors acknowledge the University of Groningen for financial support.

1. Preparation of Graphite Oxide
NOTE: Graphite oxide is prepared according to Hummers&#39; method10,11.
<ol>
	<li>Add 3 g of graphite into 69 mL of concentrated H2SO4 and stir the solution until the graphite has completely dispersed. Add 1.5 g of sodium nitrite and leave it for 1 h while stirring. Then, place the container in an ice bath.</li>
	<li>Add 9 g of potassium permanganate into the dispersion and remov...

<ol>
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It is essential that the GO layers completely cover the gold electrode wires after the deposition of GO. If the gold electrodes are not covered with GO, polyaniline will not only deposit on ERGO but also on the visible gold electrode wires directly. Deposition of polyaniline on the gold electrode wires may have implications on the performance of the electrode. After the reduction of GO to ERGO, the electrode is dried at 100 &#176;C to strengthen the bonding between the ERGO layer and the gold electrode wires. The resista...

pH plays a vital role in many chemical and biological processes. Even small changes in the pH value alter the process and adversely affect the outcome of the process. Hence, it is necessary to monitor and control the pH value during every stage of experiments. The glass-based pH electrode has been successfully used to monitor pH in many chemical and biological processes, although the use of a glass electrode poses several limitations to measuring pH. The glass-based pH electrode is relatively large, fragile, and small leakages of the electrolyte into the sample are possible. Furthermore, the electrode and electronics are relatively expensive for applications in 96-well screening fermentation systems. Moreover, the electrochemical sensors are invasive and consume the sample. Hence, it is more advantageous to use non-invasive, reference-less sensors.
Nowadays, miniaturized reaction systems are favored in many chemical engineering and biotechnology applications as these microsystems provide enhanced process control, along with many other advantages over their macro system analogs. To monitor and control the parameters in a miniaturized system is a challenging task as the sizes of the sensor to measure, for instance, pH and O2, need to be minimized as well. The successful production of microreactors for biological systems require different kinds of analytical tools for process monitoring. Hence, the development of smart microsensors plays a significant role in carrying out biological processes in microreactors.
Recently, there have been several attempts to develop smart pH sensors using chemiresistive sensing materials like carbon nanotubes and conducting polymers1. These chemiresistive sensors require no reference electrode and are easy to integrate with electronic circuits. Successful chemiresistive sensors make it possible to produce smart sensors that are cost-effective and easy to manufacture, require a small volume for testing, and are non-invasive.
Here, we report a method to develop an electrode with polyaniline-functionalized, electrochemically reduced graphene oxide. The chemiresistive electrode operates as a pH sensor during an L. lactis fermentation. L. lactis is a lactic-acid-producing bacterium used in food fermentation and food preservative processes. During fermentation, the production of lactic acid lowers the pH, and the bacterium stops growing at a low pH2,3,4.
A fermentation medium is a complex chemical environment that contains peptides, salts, and redox molecules which tend to interfere with the sensor surface5,6,7,8,9. This study shows that a pH sensor based on chemiresistive material with a proper surface protection layer could be used to measure pH in this kind of complex fermentation media. In this study, we successfully use Nafion as the protection layer for polyaniline-coated, electrochemically reduced graphene oxide to measure the pH in real-time during an L. lactis fermentation.

<table>
	<tbody>
		<tr>
			<td>Graphite flakes</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid (H2SO4)</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium nitrite (NaNO2)</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium permanganate (KMnO4)</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30 % H2O2</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HCL</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aniline</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5wt % Nafion</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>M17 powder</td>
			<td>BD Difco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphoric acid (H3PO4)</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid (HBO3)</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium dihydrogen phosphate</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dipostassium hydrogen phosphate</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Au Interdigitated electrodes</td>
			<td>BVT technology - CC1 W1</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potentiostat</td>
			<td>CH Instruments Inc (CH-600, CH-700)</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

manufacturing of a nafion-coated, reduced graphene oxide/polyaniline chemiresistive sensor to monitor ph in real-time during microbial fermentation

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). Electrochemically reduced graphene oxide acts as the conducting layer and polyaniline acts as a pH-sensitive layer. The pH-dependent conductivity of polyaniline occurs by doping of holes during protonation and by the dedoping of holes during deprotonation. We found that an ERGO-PA solid-state electrode was not functional as such in fermentation processes. The electrochemically active species that the bacteria produce during the fermentation process interfere with the electrode response. We successfully applied Nafion as a proton-conducting layer over ERGO-PA. The Nafion-coated electrodes (ERGO-PA-NA) show a good sensitivity of 1.71 &#937;/pH (pH 4 - 9) for chemiresistive sensor measurements. We tested the ERGO-PA-NA electrode in real-time in the fermentation of Lactococcus lactis. During the growth of L. lactis, the pH of the medium changed from pH 7.2 to pH 4.8 and the resistance of the ERGO-PA-NA solid-state electrode changed from 294.5 &#937; to 288.6 &#937; (5.9 &#937; per 2.4 pH unit). The pH response of the ERGO-PA-NA electrode compared with the response of a conventional glass-based pH electrode shows that reference-less solid-state microsensor arrays operate successfully in a microbiological fermentation.

The appearance of a strong reduction peak around -1.0 V (Figure 3) illustrated the reduction of GO to ERGO12,13,14,22. The intensity of the peak depends on the number of GO layers on the electrode. A thick black film completely covered the gold wires on the electrode. At that point, the two insulated gold electrodes were conductive ...

Watch this Scientific Journal Video about Manufacturing of a Nafion-coated, Reduced Graphene Oxide/Polyaniline Chemiresistive Sensor to Monitor pH in Real-time During Microbial Fermentation at JoVE.co

Manufacturing of a Nafion-coated, Reduced Graphene Oxide/Polyaniline Chemiresistive Sensor to Monitor pH in Real-time During Microbial Fermentation

Here, we report the protocol for the fabrication of a Nafion-coated, polyaniline-functionalized, electrochemically reduced graphene oxide chemiresistive micro pH sensor. This chemiresistor-based, solid-state micro pH sensor can detect pH changes in real-time during a Lactococcus lactis fermentation process.

Here, we report the engineering of a solid-state micro pH sensor based on polyaniline-functionalized, electrochemically reduced graphene oxide (ERGO-PA). ...

This technology can help us to answer key questions in a field of bioprocess technology, such as biomask conversion, or the production of biogas from organic waste. The main advantage of this chemiresistive pH sensing is that the electrodes are small, is the tube replicate in large number, and work without reference electrode. The implications of this technique extends to multiple microreactors, because pH is an important parameter.Conventional pH electrodes are too expensive or too large to be used in a microreactor screening platform. This method can provide insight into multiple permutation passes on a small scale. It can also be applied in other chemical or biological passes where pH measurements are important.First, add three grams of graphite to 69 milliliters of concentrated sulfuric acid, and stir the solution until the graphite has completely dispersed. Add 1.5 grams of sodium nitrite. After one hour of stirring, place the container in an ice bath.Add nine grams of potassium permanganate to the dispersion, and remove the container from the ice bath. After allowing the solution to warm to room temperature, add 138 milliliters of Milli-Q water drop-wise. Then add 420 milliliters of Milli-Q water and maintain the temperature at 90 degrees Celsius for 15 minutes using a hot plate.Add 7.5 milliliters of 30%hydrogen peroxide to the hot dispersion. After transferring the dispersion to a centrifuge tube, collect the product by centrifugation at 10, 000 times G for 20 minutes. After discarding the supernatant, wash the pellet four times with warm double-distilled water, and two times with 10%hydrochloric acid.Finally, wash the pellet two times with ethanol and dry it at 50 degrees Celsius in the oven. Disperse 10 milligrams of graphite oxide in 10 milliliters of Milli-Q water, and then sonicate the dispersion in an ultrasonic bath for six hours. Following ultrasonication, remove the unexfoliated graphite oxide flakes by centrifugation for 30 minutes at 2700 times G.After centrifugation, discard the solid particles, and use the supernatant for further experiments.To prepare the working solution, dilute the graphene oxide stock solution two-fold with Milli-Q water. Now add two microliters of the graphene oxide working solution onto top of an exposed interdigitated gold electrode. After drop casting, dry the graphene oxide electrode at room temperature for 12 hours.Insert the electrode in a PDMS-electrode holder. Place the other part of the electrode holder, which serves as a solution reservoir, on top of the electrode. Assemble the holders by clipping the two parts together using two paper clips.Next, pipette 300 microliters of 2 molar phosphate buffer in the reservoir. Then place the reference and counter electrodes in the solution in such a way that they are placed close to the surface of the graphene oxide film. Connect the electrodes with a potentiostat, connected to a computer for data acquisition.Use cyclic voltammetry for the electrochemical reduction, and select the appropriate potential range and scan rate. Cycle the voltage over the electrode 10 times between zero to minus 1.2 volts. After the experiment, remove the electrode from the holder, and repeatedly wash it with Milli-Q water.Then dry the electrode in an oven at 101 degrees Celsius for 12 hours. When the electrode is dry, remove it from the oven and allow it to cool down to room temperature. Then measure the conductivity of the ErGO electrode with a multimeter.Prepare a 10 millimolar solution of aniline monomer for the polyaniline functionalization, by dissolving five microliters of 10 millimolar aniline in five milliliters of one molar sulfuric acid. Add 300 microliters of aniline monomer to the solution reservoir. Then place the ErGO deposited electrode into the electrode holder as previously described.Use cyclic voltammetry for the electropolymerization of aniline to functionalize the ErGO into ErGO-PA, and select the appropriate potential range and scan rate. Cycle the voltage over the electrode 50 times, between zero to 9 volts. After the polyaniline deposition, remove the electrode and repeatedly wash it with Milli-Q water.Then dry the electrode at 80 degrees Celsius in the oven for 12 hours. When the electrode is dry, remove it from the oven and allow it to cool down to room temperature before measuring the conductivity of the electrode with a multimeter. Next, add 2 molar sodium hydroxide to Britton-Robinson buffer solution, until the pH is five.To prepare a Britton-Robinson universal buffer solution, mix 04 moles of phosphoric acid, 04 moles of acetic acid, and 04 moles of boric acid, in 8 liters of Milli-Q water. Then, add Milli-Q water until the final volume is one liter. After conditioning the electrode in the pH five buffer solution, measure its resistance in solutions of different pH, by first dipping it directly in the buffer solution.Then connect the other part of the electrode to a computer-controlled potentiostat for data acquisition. Choose amperometry current versus time curve from the list of techniques, and apply a 100 millivolt potential difference to the electrode. After the measurements, dry the electrode at room temperature for 12 hours.Add five microliters of five weight-percent nafion on top of the ErGO-PA electrode, and dry the electrode at room temperature for 12 hours. After the nafion coating, heat the electrode in the buffer solution at pH five for 24 hours before pH measurements. After conditioning in the pH five buffer solution, remove the nafion-coated ErGO-PA electrode and measure the resistance of the electrode from pH four to nine, as previously described.Add 9.3 grams of M17 powder to 250 milliliters of demineralized water. Slowly agitate the solution until the powder dissolves completely. Then autoclave the solution at 121 degrees Celsius for 15 minutes.Next, add 50 milliliters of the sterilized M17 medium in a 250 milliliter sterilized flask with a magnetic stir bar. Add 8 milliliters of autoclaved 1 molar glucose solution to the medium. Then, inoculate the solution with 10 microliters of L.lactis culture, previously grown in the same culture medium.Place the flask with the inoculated culture medium on a magnetic stir plate in an incubation oven at 30 degrees Celsius for 18 hours while stirring. Monitor the pH during incubation. Place the ErGO-PA-NA electrode into the L.lactis culture and close it with a cotton plug.Then place the set up into a thermostat at 30 degrees Celsius to grow L.lactis. Following this, apply 100 millivolts to the electrode and measure the current against time. Take 5 milliliter samples at different time points to measure offline the optical density at 600 nanometers, and the pH with a conventional glass electrode.Continue the measurements until the optical density of the culture becomes constant, indicating that the bacteria are not growing anymore. The appearance of a strong reduction peak around minus 1.0 volts illustrated the reduction of graphene oxide to ErGO. The intensity of the peak depends on the number of graphene oxide layers on the electrode.When the ErGO-PA electrode was placed in pH four to nine buffer solutions, the current increased with increasing pH due to the doping and de-doping of holes during the protonation deprotonation process. The response of the electrode was immediately stable when the addition of sodium hydroxide stopped at a particular pH. The conductivity of the electrode was not affected much by the nafion coating.But a few ohms of difference in the resistance value occurred, and changed the base current value of the ErGO-PA electrode. Similar to the ErGO-PA electrode, the resistance of the ErGO-PA-NA electrode changed when the pH of the buffer solution changed from four to nine. Once the growth of L.lactis started, the current of the ErGO-PA-NA electrode decreased gradually, and then accelerated during the exponential growth phase, reaching a stable value at the end of the growth.The final value of the current is comparable to the current value of the ErGO-PA-NA electrode tested in buffer solution. While attempting this procedure, it is important to remember to completely cover the gold electrodes with the graphene oxide. This technology paved the way for researchers in the field of process control, to manufacture and use small pH electrodes for biological and chemical systems.Don't forget that working with concentrated sulfuric acid, potassium permanganate, and hydrogen peroxide can be extremely hazardous. This procedure must be performed in a fume hood.

manufacturing-nafion-coated-reduced-graphene-oxidepolyaniline

Research

JoVE Journal

Bioengineering

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Simultaneous Synthesis of Single-walled Carbon Nanotubes and Graphene in a Magnetically-enhanced Arc Plasma

Carbon nanostructures such as single-walled carbon nanotubes (SWCNT) and graphene attract a deluge of interest of scholars nowadays due to their very promising application for molecular sensors, field effect transistor and super thin and flexible electronic devices1-4. Anodic arc discharge supported by the erosion of the anode material is one of the most practical and efficient methods, which can provide specific non-equilibrium processes and a high influx of carbon material to the developing structures at relatively higher temperature, and consequently the as-synthesized products have few structural defects and better crystallinity.
 To further improve the controllability and flexibility of the synthesis of carbon nanostructures in arc discharge, magnetic fields can be applied during the synthesis process according to the strong magnetic responses of arc plasmas. It was demonstrated that the magnetically-enhanced arc discharge can increase the average length of SWCNT 5, narrow the diameter distribution of metallic catalyst particles and carbon nanotubes 6, and change the ratio of metallic and semiconducting carbon nanotubes 7, as well as lead to graphene synthesis 8. 
 Furthermore, it is worthwhile to remark that when we introduce a non-uniform magnetic field with the component normal to the current in arc, the Lorentz force along the J&times;B direction can generate the plasmas jet and make effective delivery of carbon ion particles and heat flux to samples. As a result, large-scale graphene flakes and high-purity single-walled carbon nanotubes were simultaneously generated by such new magnetically-enhanced anodic arc method. Arc imaging, scanning electron microscope (SEM), transmission electron microscope (TEM) and Raman spectroscopy were employed to analyze the characterization of carbon nanostructures. These findings indicate a wide spectrum of opportunities to manipulate with the properties of nanostructures produced in plasmas by means of controlling the arc conditions.

Anodic arc discharge is one of the most practical and efficient methods to synthesize various carbon nanostructures. To increase the arc controllability and flexibility, a non-uniform magnetic field was introduced to process the one-step synthesis of large-scale graphene flakes and high-purity single-walled carbon nanotubes.

Carbon nanostructures such as single-walled carbon nanotubes (SWCNT) and graphene attract a deluge of interest of scholars nowadays due to their very ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to investigate mechanisms governing cell adhesion under dynamic flow conditions. For instance, cancer cells labeled with multiple fluorophores can be perfused over a potentially reactive substrate to model mechanisms of cancer metastasis. However, multi-channel single camera systems and color cameras exhibit shortcomings in image acquisition for real-time live cell analysis. To overcome these limitations, we used a dual camera emission splitting system to simultaneously capture real-time image sequences of fluorescently labeled cells in the flow chamber. Dual camera emission splitting systems filter defined wavelength ranges into two monochrome CCD cameras, thereby simultaneously capturing two spatially identical but fluorophore-specific images. Subsequently, psuedocolored one-channel images are combined into a single real-time merged sequence that can reveal multiple target molecules on cells moving rapidly across a region of interest.

Dual camera emission splitting systems for two-color fluorescence microscopy generate real-time image sequences with exceptional optical and temporal resolution, a requirement of certain live cell assays including parallel plate flow chamber adhesion assays. When software is employed to merge images from simultaneously acquired emission channels, pseudocolored image sequences are produced.

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic performance of microalgae-based biofuel production. Models that estimate microalgal growth under different conditions can help to optimize PBR design and operation. To be effective, the growth parameters used in these models must be accurately determined. Algal growth experiments are often constrained by the dynamic nature of the culture environment, and control systems are needed to accurately determine the kinetic parameters. The first step in setting up a controlled batch experiment is live data acquisition and monitoring. This protocol outlines a process for the assembly and operation of a bench-scale photosynthetic bioreactor that can be used to conduct microalgal growth experiments. This protocol describes how to size and assemble a flat-plate, bench-scale PBR from acrylic. It also details how to configure a PBR with continuous pH, light, and temperature monitoring using a data acquisition and control unit, analog sensors, and open-source data acquisition software.

This paper describes the assembly process and operation of a bench-scale photosynthetic bioreactor that can be used, in conjunction with other methods, to estimate pertinent kinetic growth parameters. This system continuously monitors the pH, light, and temperature using sensors, a data acquisition and control unit, and open-source data acquisition software.

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved oxygen concentration). Nevertheless, the morphology of cells can be a suitable indicator for optimal conditions, since it changes with dependence on the growth state, product accumulation and cell stress. Furthermore, the single-cell size distribution provides not only information about the cultivation conditions, but also about the population heterogeneity. To gain such information, a photo-optical in situ microscopy device1 was developed to enable the monitoring of the single-cell size distribution directly in the cell suspension in bioreactors. An automated image analysis is coupled to the microscopy based on a neural network model, which is trained with user-annotated images. Several parameters, which are gained from the captures of the microscope, are correlated to process relevant features of the cells, like their metabolic activity. Until now, the presented in situ microscopy probe series was applied to measure the pellet size in filamentous fungi suspensions. It was used to distinguish the single-cell size in microalgae cultivation and relate it to lipid accumulation. The shape of cellular particles was related to budding in yeast cultures. The microscopy analysis can be generally split into three steps: (i) image acquisition, (ii) particle identification, and (iii) data analysis, respectively. All steps have to be adapted to the organism, and therefore specific annotated information is required in order to achieve reliable results. The ability to monitor changes in cell morphology directly in line or on line (in a by-pass) enables real-time values for monitoring and control, in process development as well as in production scale. If the off line data correlates with the real-time data, the current tedious off line measurements with unknown influences on the cell size become needless.

In Situ Microscopy for Real-time Determination of Single-cell Morphology in Bioprocesses

A photo-optical in situ microscopy device was developed to monitor the size of single cells directly in the cell suspension. The real-time measurement is conducted by coupling the photo-optical sterilizable probe to an automated image analysis. Morphological changes appear with dependence on the growth state and cultivation conditions.

In situ monitoring in microbial bioprocesses is mostly restricted to chemical and physical properties of the medium (e.g.,pH value and the dissolved ...

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread chondrocyte death, potentially leading to irreversible breakdown of the joint and the onset of osteoarthritis. Additionally, maintenance of chondrocyte viability is important in osteochondral graft procedures for optimal surgical outcomes. We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine synovial joints after application of controlled mechanical loads or impacts. This method can be used in comparative studies to investigate the effects of different mechanical loading regimens, different environmental conditions or genetic manipulations, as well as different stages of cartilage degeneration on short- and/or long-term vulnerability of in situ articular chondrocytes. The goal of the protocol introduced in the manuscript is to assess the spatial extent of cell injury/death on the articular surface of murine synovial joints. Importantly, this method enables testing on fully intact cartilage without compromising native boundary conditions. Moreover, it allows for real-time visualization of vitally stained articular chondrocytes and single image-based analysis of cell injury induced by application of controlled static and impact loading regimens. Our representative results demonstrate that in healthy cartilage explants, the spatial extent of cell injury depends sensitively on load magnitude and impact intensity. Our method can be easily adapted to investigate the effects of different mechanical loading regimens, different environmental conditions or different genetic manipulations on the mechanical vulnerability of in situ articular chondrocytes.

We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine joints after application of controlled mechanical loads or impacts. This method can be used to investigate how osteoarthritis, genetic factors and/or different loading regimens affect the vulnerability of in situ chondrocytes.

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread ...

Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. Microfluidic cancer-on-chip models have been proposed to bridge the gap between the oversimplified conventional 2D cultures and more complicated animal models, which have limited ability to produce reliable and reproducible quantitative results. Here, we present a microfluidic cancer-on-chip model that reproduces key components of a complex tumor microenvironment in a comprehensive manner, yet is simple enough to provide robust quantitative descriptions of cancer dynamics. This microfluidic cancer-on-chip model, the &#34;Evolution Accelerator,&#34; breaks down a large population of cancer cells into an interconnected array of tumor microenvironments while generating a heterogeneous chemotherapeutic stress landscape. The progression and the evolutionary dynamics of cancer in response to drug gradient can be monitored for weeks in real time, and numerous downstream experiments can be performed complementary to the time-lapse images taken through the course of the experiments.

We present a microfluidic cancer-on-chip model, the &#34;Evolution Accelerator&#34; technology, which provides a controllable platform for long-term real-time quantitative studies of cancer dynamics within well-defined environmental conditions at the single-cell level. This technology is expected to work as an in vitro model for fundamental research or pre-clinical drug development.

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. ...