The authors of this paper do not have any competing financial interests.

1. PEDOT:PSS Solution Preparation
<ol>
	<li>To 50 ml&#160;of PEDOT:PSS, add ethylene glycol (increases conductivity) in a volume ratio of 1:4 (ethylene glycol to PEDOT:PSS), 0.5 &#956;l/ml&#160;of Dodecylbenzenesulfonic acid (DBSA) as a surfactant, and 10 mg/ml 3-glycidoxypropyltrimethoxysilane (GOPS) as a cross-linker to promote adhesion of the conducting polymer to the glass slide.</li>
</ol>
2. OECT Fabrication (Figure 3)
<ol>
	<li>Define thermally evaporated gold source and drain contacts via lift-off lithography:
	<ol>
		<li>Spin coat photoresist on a normal clean glass slide at 3,000 rpm for 30 sec. Glass dimensions are 3 in&#160;x 1 in.</li>
		<li>Define patterns by photolithography. Dimensions may be altered, however the dimensions listed here were shown to lead to the optimal sensitivity. Then use a developer.</li>
		<li>Evaporate 5 nm and 100 nm of chromium and gold, respectively.</li>
		<li>Lift-off the photoresist in an acetone bath for 1 hr, leaving the substrate with the source and drain Au contacts area only.</li>
	</ol>
	</li>
	<li>The desired length of the PEDOT:PSS channel is 1 mm. This is achieved by patterning using a parylene-C (Pa-C) peel-off technique:
	<ol>
		<li>Load 3.5 g&#160;of Pa-C in the coating setup. Uniformly evaporate 2 &#181;m of parylene-C on top of the substrate with Au contacts.</li>
		<li>Pattern the channel by photolithography:
		<ol>
			<li>Spin coat photoresist at 3,000 rpm for 30 sec.</li>
			<li>Use a mask to illuminate channel and gold contact 20 sec to UV, the exposed photoresist will become soluble in the developer.</li>
			<li>Use developer to open the channel area on the photoresist.</li>
		</ol>
		</li>
		<li>Etch the Pa-C in the channel area by a 15 min plasma step (O2 (50 sccm) and CHF4 (3 sccm) plasma at 160 W) in order to open the channel and the gold contact.</li>
	</ol>
	</li>
	<li>Deposit the PEDOT:PSS mixture solution by spin coating at 500 rpm for 45 sec. Bake for 30 sec at 110 &#176;C.</li>
	<li>Peel-off the Pa-C to reveal the PEDOT:PSS channel of the substrate underneath. This channel should slightly overlap with the Au contacts made in Protocol&#160;1.</li>
	<li>Bake samples for 1 hr at 140 &#176;C under atmospheric conditions.</li>
</ol>
3. Device Assembly
<ol>
	<li>Make PDMS by mixing two solutions (usually supplied together in a kit) at a ratio 1:10 of curing solution in base solution. Bake it for 1 hr at 120 &#176;C.</li>
	<li>Design well by using a hole punch and cut a square around the desired area.</li>
	<li>Glue a PDMS well on top of the channel, to result in a channel area of approximately 6 mm2. Ensure that the source and drain contacts are not covered.</li>
	<li>Make a plastic support with a hole in it (can use the holder for the cell culture insert) and then glue it on top of the PDMS. Let it dry overnight.</li>
	<li>Test the well for leaking by prefilling with water.</li>
	<li>Solder electric wires on the source and drain contact with tin.</li>
</ol>
4. Cell Culture
<ol>
	<li>Prepare cell culture media as follows:
	<ol>
		<li>In DMEM, add 1% Glutamine, 10% Fetal Bovine Serum, 0.5% Penicillin-streptomycin, and 0.1% Gentamicin.</li>
		<li>Sterilize the cell culture media using a sterile filter device.</li>
	</ol>
	</li>
	<li>Maintain Caco-2 cells between passage 49 and 68 at 37 &#176;C in a humidified atmosphere of 5% CO2, in cell culture media.</li>
	<li>Divide cells once a week using trypsin and seed at 1.5 x 104 cells/insert.</li>
	<li>Change cell culture media twice a week over 3 weeks.</li>
</ol>
5. Measurements with OECT
<ol>
	<li>Connect an Ag/AgCl wire to a sourcemeter for use as the gate electrode. Immerse the tip in cell culture media, which is used as the electrolyte, as illustrated in Figure 1a.</li>
	<li>Connect wires from source and drain to the sourcemeter (dual channel setup).</li>
	<li>Apply a square pulse positive voltage (VGS) between the gate and the source (used as reference electrode: ground). A negative constant voltage between drain and source (VDS) is applied and corresponding current (IDS) is measured.</li>
	<li>Use a PC running program data collection. OECT parameters entered in a customized acquisition program should be as follows: VDS = -0.2 V, VGS = 0.3 V, VGS on time = 2 sec, off time = 28 sec.</li>
	<li>Read out source-drain current (IDS) and gate current (IGS). Carry out the measurement for several minutes to ensure a stable baseline signal.</li>
</ol>
6. Integrating Cells with OECT for Measurement
Note: Prior to the experiment, the integrity of the cell layer may be verified by measuring TER with an Impedance spectroscopy device. TER of each 3-week old Caco-2 cell insert should be above 400&#160;&#937;.cm2.
<ol>
	<li>During off time of OECT measurement: Remove the gate electrode from the electrolyte, incorporate the cell culture insert, and replace the gate electrode inside the cell culture insert.</li>
	<li>Carry out a baseline measurement with the cells for several minutes to ensure a stable signal. This baseline will be used as for calculation of the normalized value corresponding to an intact monolayer (0).</li>
</ol>
7. Preparation and Introduction of Toxic Compound
<ol>
	<li>Prepare a 1 M solution of EGTA in DI water, first adjusting the pH of the solution to 7.4 with Tris base.</li>
	<li>Add EGTA solution in the appropriate volume to obtain the desired concentration (e.g. between 5 mM and 100 mM) taking into account the&#160;volume. The EGTA should be added in the basal chamber during the off time.</li>
	<li>Measure continuously for 90 min as in Protocol 5. If the measurement is being carried out at room temperature, the stability of the cells will not remain constant after 90 min.</li>
	<li>At the end of the run, scratch the cell layer to result in a complete destruction of the barrier layer and measure for 15 min, this point could be done by removing the filter and immersing the gate electrode in the media. This baseline will be used as for calculation of the normalized value corresponding to a destroyed monolayer.</li>
</ol>

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This project is supported by FP7-People-2009-RG, Marie Curie Project No. 256367 (CELLTOX) and the European Research Council ERC-2010-StG Proposal No 258966 (IONOSENSE), as well as a joint grant from the Conseil Regional de Provence Alpes Cote d'Azur and CDL Pharma for ST. We thank George Malliaras for useful discussions pertaining to OECT design and function.

This technique provides a novel method to integrate an organic electrochemical transistor with live cells to measure barrier tissue integrity. The main advantages of this technique are the rapidity and sensitivity, but also the low cost of the device for dynamic monitoring of barrier tissue.
As this method uses live cells, a critical point is to be sure to use a monolayer, which represents an intact barrier layer. The parameters of the barrier should be defined during the characterization of t...

The gastrointestinal epithelium is an example of barrier tissue, which controls the passage of molecules between different compartments of the body. The epithelium consists of elongated columnar cells joined together by complexes of proteins that provide a physical barrier1 against pathogens and toxins, while allowing the passage of water and nutrients required to sustain the body. This selectivity is due to the polarization of the epithelial cells, which creates two different membrane domains: the apical side of the cells exposed to the lumen and the basal side of the cells anchored on the underlying tissues2,3. Tight junctions (TJ) are complexes of proteins present at the apical portion of epithelial cells and are part of a larger complex known as the apical junction4. Ion flow across the barrier tissue may either go via the transcellular (through the cell) or via a paracellular (between two adjacent cells) pathway. The sum of the transport through both pathways is known as the transepithelial resistance. The apical junction is responsible for the regulation of ions and molecules passing across the barrier5,6 via a specific opening and closing function. A dysfunction or disruption of these protein complexes is often related to disease7-11. In addition, many enteric pathogens/toxins are known to specifically target this complex, thereby entering the body and leading to diarrhea, most likely as a consequence of massive dysregulation of ion/water flow across the barrier12-14. Barrier tissue may also be modified by changing the extracellular microenvironment. Cadherin is a critical protein for cell-cell adhesion, and is involved in the formation of the apical junction. Calcium is required for the correct structural conformation of Cadherin, and a decrease in extracellular calcium has been shown to result in the destruction of the cell-cell junction and a subsequent opening of the paracellular pathway between the cells15. In this study, EGTA (Ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid), a specific calcium chelator, was used to induce a breach in barrier tissue, as it has been shown to have a rapid and drastic effect on paracellular ion flow16,17. This calcium chelator was used on a confluent and differentiated monolayer of the Caco-2 cell-line. Cultured in cell culture inserts, this cell line is known to develop the characteristics of the gastrointestinal tract and is widely used by the pharmaceutical industry to test the absorption of drugs18,19.
Methods to monitor barrier tissue integrity are plentiful. These methods are often optical, relying on immunofluorescence staining of particular proteins known to be at the apical junction20, or relying on the quantification of a fluorescent tracer molecule that is normally impermeable to the barrier tissue21,22. However, label-free methods (i.e. without a fluorophore/chromophore) are preferable as the use of a label can incur artifacts, and often increases cost and assay time. Electrical, label-free monitoring of barrier tissue&#160;has recently emerged as a dynamic monitoring method23. For example recent technological advances in electrical impedance spectroscopy have allowed the development of a commercially available scanning apparatus24,25 that can measure transepithelial resistance (TER), a measurement of the ion conductance across the cell layer.
Organic electronics has created a unique opportunity to interface the world of electronics and biology26,27 28,29 by using conducting polymers that&#160;can conduct both electronic and ionic carriers. A new technique to detect breaches in barrier tissue using the OECT30-32 was recently introduced. This device was validated against existing techniques used to assess barrier tissue integrity, including immunofluorescence, permeability assays using Lucifer yellow, and impedance spectroscopy using the Cellzscope. In the case of all toxic compounds tested, the OECT was found to operate with equal or better sensitivity, and with increased temporal resolution, compared to the above techniques. In this device, PEDOT:PSS, a conducting polymer that has been shown to be stable and biocompatible33,34, is used as the active material in the transistor channel. The OECT is composed of drain and source electrodes on either side of a conducting polymer channel. This is then placed in contact with an electrolyte, which forms an integral part of the device. A gate electrode is immersed in the electrolyte (Figure 1), and when a positive gate voltage is applied at the gate, cations from the electrolyte are forced into the channel, thus dedoping the conducting polymer and resulting in a change in the source-drain current. The device is thus extremely sensitive to minute changes in ionic flux due to amplification by the transistor. A cell layer grown on a cell culture insert was placed between the gate electrode and the conducting polymer channel. The presence of an intact cell layer acts as barrier for the cations entering into the conducting polymer, therefore, in the presence of an intact monolayer, the drain current decreases (Figure 2: transition from region a to b). In the presence of a toxic compound, the barrier tissue will progressively lose its integrity, letting the cations enter into the polymer film and increasing the drain current (Figure 2: region c). With this technique, the breach in barrier tissue is seen by the modulation of the drain current, corresponding to the modulation of the flux across the monolayer. This device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity in real time. This technology will be of interest in the domain of toxicology for drug testing, disease diagnostics or basic research as the barrier model can be easily adapted. This method will also help to reduce animal experimentation, as it allows the validation of in vitro models to replace in vivo testing.

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			<td height="43" style="height:43px;width:216px;">CLEVIOS PH 1000</td>
			<td style="width:87px;">HERAUS CLEVIOS</td>
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			<td style="width:167px;"></td>
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		<tr height="64">
			<td height="64" style="height:64px;width:216px;">AZ9260 resin</td>
			<td style="width:87px;">CIPEC SPECIALITIES</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Dodecylbenzenesulfonic acid (DBSA)</td>
			<td style="width:87px;">Acros Organic</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">3-glycidoxypropyltrimethoxysilane (GOPS)</td>
			<td style="width:87px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">24-well Suspended cell Culture insert Millicell&#160; PET 0.4 &#956;m Millipore</td>
			<td style="width:87px;">Dominique dutscher</td>
			<td style="width:119px;">51705</td>
			<td style="width:167px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">24-well cell culture plate BD Falcon</td>
			<td style="width:87px;">Dominique dutscher</td>
			<td style="width:119px;">51705</td>
			<td style="width:167px;"></td>
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		<tr height="43">
			<td height="43" style="height:43px;width:216px;">STERICUP-GP PES 0.22 &#956;M</td>
			<td style="width:87px;">Dominique dutscher</td>
			<td style="width:119px;">51246</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">ADVANCED DMEM Marque GIBCO</td>
			<td style="width:87px;">Fisher scientific</td>
			<td style="width:119px;">E3434T</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">FBS HEAT INACT. S.AMERICAN</td>
			<td style="width:87px;">Fisher scientific</td>
			<td style="width:119px;">E3387M</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">PENICILLIN STREPTOMYCIN</td>
			<td style="width:87px;">Fisher scientific</td>
			<td style="width:119px;">E3470C</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">GLUTAMAX</td>
			<td style="width:87px;">Fisher scientific</td>
			<td style="width:119px;">E3524T</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">TRYPSIN 0.05% EDTA</td>
			<td style="width:87px;">Fisher scientific</td>
			<td style="width:119px;">E3513N</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid)</td>
			<td style="width:87px;">Sigma Aldrich</td>
			<td style="width:119px;">E4378</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">ETHYLENE GLYCOL, ANHYDROUS, 99.8%,</td>
			<td style="width:87px;">Sigma aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Caco-2 cells</td>
			<td style="width:87px;">ATCC</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">PDMS</td>
			<td style="width:87px;">Dow corning</td>
			<td style="width:119px;">SYLGARD 184 SILICONE ELASTOMER</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Au (99.99%)</td>
			<td style="width:87px;">NEYCO</td>
			<td style="width:119px;">AU3X6</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Chromium (99.95%)</td>
			<td style="width:87px;">NEYCO</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
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			<td height="64" style="height:64px;width:216px;">Parylene C</td>
			<td style="width:87px;">Specialty Coating Systems</td>
			<td style="width:119px;"></td>
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			<td height="43" style="height:43px;width:216px;">Ag/AgCl wire</td>
			<td style="width:87px;">HARVARD APPARATUS</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
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			<td height="64" style="height:64px;width:216px;">Photoresist</td>
			<td style="width:87px;">CIPEC SPECIALITIES</td>
			<td style="width:119px;">R&#233;sine AZ9260</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
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sensing of barrier tissue disruption with an organic electrochemical transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

During the first step of measurement, the drain current may vary somewhat, but in most cases it should remain stable (Figure 2, section a). If the signal is not stable, the transistor should be discarded and replaced. This stability check also ensures that any initial losses in conductivity of the device do not affect the subsequent measurement. After several minutes of measurement, the insert with cells forming barrier tissue is placed on top of the channel. The drain current should immediately decrease...

Watch this Scientific Journal Video about Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor at JoVE.com

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

sensing-barrier-tissue-disruption-with-an-organic-electrochemical

Research

JoVE Journal

Bioengineering

11.6K Views.  Ecole Nationale Superieure des Mines. The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

Video: Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

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 ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

Preparation of Silicon Nanowire Field-effect Transistor for Chemical and Biosensing Applications

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we describe the preparation of polycrystalline silicon nanowire field-effect transistors (pSNWFETs) that serve as biosensing devices for biomarker detection. A protocol for chemical and biomolecular sensing by using pSNWFETs is presented. The pSNWFET device was demonstrated to be a promising transducer for real-time, label-free, and ultra-high-sensitivity biosensing applications. The source/drain channel conductivity of a pSNWFET is sensitive to changes in the environment around its silicon nanowire (SNW) surface. Thus, by immobilizing probes on the SNW surface, the pSNWFET can be used to detect various biotargets ranging from small molecules (dopamine) to macromolecules (DNA and proteins). Immobilizing a bioprobe on the SNW surface, which is a multistep procedure, is vital for determining the specificity of the biosensor. It is essential that every step of the immobilization procedure is correctly performed. We verified surface modifications by directly observing the shift in the electric properties of the pSNWFET following each modification step. Additionally, X-ray photoelectron spectroscopy was used to examine the surface composition following each modification. Finally, we demonstrated DNA sensing on the pSNWFET. This protocol provides step-by-step procedures for verifying bioprobe immobilization and subsequent DNA biosensing application.

We describe key steps for biosensing by using polysilicon nanowire field-effect transistors, including the preparation of the device and the immobilization and confirmation of a DNA molecular probe on the nanowire surface, as well as conditions for DNA sensing.

Surveillance using biomarkers is critical for the early detection, rapid intervention, and reduction in the incidence of diseases. In this study, we ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Wet Chemistry and Peptide Immobilization on Polytetrafluoroethylene for Improved Cell-adhesion

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly interesting for already approved polymers that have a long standing use in medicine because these materials are well characterized and legal issues associated with the introduction of newly synthesized polymers may be avoided. Polytetrafluoroethylene (PTFE) is one of the most frequently employed materials for the manufacturing of vascular grafts but the polymer lacks cell adhesion promoting features. Endothelialization, i.e., complete coverage of the grafts inner surface with a confluent layer of endothelial cells is regarded key to optimal performance, mainly by reducing thrombogenicity of the artificial interface.
This study investigates the growth of endothelial cells on peptide-modified PTFE and compares these results to those obtained on unmodified substrate. Coupling with the endothelial cell adhesive peptide Arg-Glu-Asp-Val (REDV) is performed via activation of the fluorin-containing polymer using the reagent sodium naphthalenide, followed by subsequent conjugation steps. Cell culture is accomplished using Human Umbilical Vein Endothelial Cells (HUVECs) and excellent cellular growth on peptide-immobilized material is demonstrated over a two-week period.

Cell-adhesiveness is key to many approaches in biomaterial research and tissue engineering. A step-by-step technique is presented using wet-chemistry for the surface modification of the important polymer PTFE with peptides.

Endowing materials surface with cell-adhesive properties is a common strategy in biomaterial research and tissue engineering. This is particularly ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

In Vitro Multiparametric Cellular Analysis by Micro Organic Charge-modulated Field-effect Transistor Arrays

Modern electrophysiology has been constantly fueled by the parallel development of increasingly sophisticated tools and materials. In turn, discoveries in this field have driven technological progress in a back-and-forth process that ultimately determined the impressive achievements of the past 50 years. However, the most employed devices used for cellular interfacing (namely, the microelectrode arrays and microelectronic devices based on transistors) still present several limitations such as high cost, the rigidity of the materials, and the presence of an external reference electrode. To partially overcome these issues, there have been developments in a new scientific field called organic bioelectronics, resulting in advantages such as lower cost, more convenient materials, and innovative fabrication techniques.
Several interesting new organic devices have been proposed during the past decade to conveniently interface with cell cultures.&#160;This paper presents the protocol for the fabrication of devices for cellular interfacing based on the organic charge-modulated field-effect transistor (OCMFET). These devices, called micro OCMFET arrays (MOAs), combine the advantages of organic electronics and the peculiar features of the OCMFET to prepare transparent, flexible, and reference-less tools with which it is possible to monitor both the electrical and the metabolic activities of cardiomyocytes and neurons in vitro, thus allowing a multiparametric evaluation of electrogenic cell models.

In Vitro Multiparametric Cellular Analysis by Micro Organic Charge-modulated Field-effect Transistor Arrays

Here, we present the fabrication protocol of an organic charge-modulated field-effect transistor (OCMFET)-based device for in vitro cellular interfacing. The device, called a micro OCMFET array, is a flexible, low-cost, and reference-less device, which will enable the monitoring of the electrical and metabolic activities of electroactive cell cultures.

Modern electrophysiology has been constantly fueled by the parallel development of increasingly sophisticated tools and materials. In turn, discoveries in ...

Disruption of the Blood-Spinal Cord Barrier Using Low-Intensity Focused Ultrasound in a Rat Model

Low-intensity focused ultrasound (LIFU) uses ultrasonic pulsations at lower intensities than ultrasound and is being tested as a reversible and precise neuromodulatory technology. Although LIFU-mediated blood-brain barrier (BBB) opening has been explored in detail, no standardized technique for blood-spinal cord barrier (BSCB) opening has been established to date. Therefore, this protocol presents a method for successful BSCB disruption using LIFU sonication in a rat model, including descriptions of animal preparation, microbubble administration, target selection&#160;and localization, as well as BSCB disruption visualization and confirmation. The approach reported here is particularly useful for researchers who need a fast and cost-effective method to test and confirm target localization and precise BSCB disruption in a small animal model with a focused ultrasound transducer, evaluate the BSCB efficacy of sonication parameters, or explore applications for LIFU at the spinal cord, such as drug delivery, immunomodulation, and neuromodulation. Optimizing this protocol for individual use is recommended, especially for advancing future preclinical, clinical, and translational work.

Disruption of the blood-spinal cord barrier (BSCB) can be successfully achieved with the intravenous administration of microbubbles and the application of low-intensity focused ultrasound (LIFU). This protocol details the opening of the BSCB using LIFU in a rodent model, including equipment setup, microbubble injection, target localization, and BSCB disruption visualization.

Low-intensity focused ultrasound (LIFU) uses ultrasonic pulsations at lower intensities than ultrasound and is being tested as a reversible and precise ...

Evaluating MOF-Induced Cell Behavior Changes by Electric Cell-Substrate Sensing

Electric Cell-Substrate Sensing for Real-Time Evaluation of Metal-Organic Framework Toxicological Profiles

Metal-organic frameworks (MOFs) are hybrids formed through the coordination of metal ions and organic linkers in organic solvents. The implementation of MOFs in biomedical and industrial applications has led to concerns regarding their safety. Herein, the profile of a selected MOF, a zeolitic imidazole framework, was evaluated upon exposure to human lung epithelial cells. The platform for evaluation was a real-time technique (i.e., electric cell-substrate impedance sensing [ECIS]). This study identifies and discusses some of the deleterious effects of the selected MOF on the exposed cells. Furthermore, this study demonstrates the benefits of using the real-time method versus other biochemical assays for comprehensive cell evaluations. The study concludes that observed changes in cell behavior could hint at possible toxicity induced upon exposure&#160;to MOFs of different physicochemical characteristics and the dosage of those frameworks being used. By understanding changes in cell behavior, one foresees the ability to improve safe-by-design strategies of MOFs to be used for biomedical applications by specifically tailoring their physicochemical characteristics.

Author Spotlight: Advances in Evaluating Human Lung Epithelial Cells&#039; Response to Metal-Organic Frameworks 

The following study evaluates the toxicological profile of a selected metal-organic framework utilizing electric cell-substrate impedance sensing (ECIS), a real-time, high-throughput screening technique.

Metal-organic frameworks (MOFs) are hybrids formed through the coordination of metal ions and organic linkers in organic solvents. The implementation of ...