Name: merging ion concentration polarization between juxtaposed ion exchange membranes to block the propagation of the polarization zone
Uploaded: 2017-02-23T14:00:00
Description: Watch this Scientific Journal Video about Merging Ion Concentration Polarization between Juxtaposed Ion Exchange Membranes to Block the Propagation of the Polarization Zone at JoVE.com

This work was supported by the internal fund of the Korea Institute of Science and Technology (2E26180) and by the Next Generation Biomedical Device Platform program, funded by the National Research Foundation of Korea (NRF-2015M3A9E202888).

1. Fabrication of Cation Exchange Membrane-integrated Microfluidic Chips
<ol>
	<li>Preparation of silicon masters
	<ol>
		<li>Design two kinds of silicon masters: one for patterning a cation exchange resin and the other for building a microchannel with polydimethylsiloxane (PDMS). 
		NOTE: The detail geometry will be described in the steps 1.3.1 and 1.4.1.</li>
		<li>Fabricate the silicon masters by using either conventional photolithography or deep reactive ion etching27.<...

<ol>
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We have described the fabrication protocol and the performance of a spatiotemporally defined preconcentrator in a range of the applied voltage (0.5-100 V), ionic strength (1-100 mM), and pH (3.7-10.3), achieving a 10,000-fold preconcentration of dyes and protein within 10 min. As like previous ICP devices, the preconcentration performance becomes better at higher voltage and at lower ionic strength. One additional parameter we can consider here is the distance between two cation exchange membranes. If we increase the int...

Ion concentration polarization (ICP) refers to a phenomenon that occurs during ion enrichment and ion depletion on a permselective membrane, resulting in an additional potential drop with ion concentration gradients1,2. This concentration gradient is linear, and it becomes steeper as a higher voltage is applied (Ohmic regime) until the ion concentration on the membrane approaches zero (limiting regime). At this diffusion-limited condition, the gradient (and corresponding ion flux) has been known to be maximized/saturated1. Beyond this conventional understanding, when the voltage (or current) is increased further, an overlimiting current is observed, with flat depletion zones and very sharp concentration gradients at the zone boundary1,3. The flat zone has a very low ion concentration, but surface conduction, electro-osmotic flow (EOF), and/or electro-osmotic instability promote ion flux and induce an overlimiting current3,4,5. Interestingly, the flat depletion zone serves as an electrostatic barrier, which filters out6,7,8,9 and/or preconcentrates targets10,11. Since there is an insufficient amount of ions to screen the surface charges of charged particles (for satisfying electroneutrality), the particles cannot pass through this depletion zone and therefore line up at its boundary. This nonlinear ICP effect is a generic phenomenon in various types of membranes10,11,12,13,14 and geometries6,15,16,17,18,19,20,21; this is why researchers have been able to develop various types of filtration6,7,8,9 and preconcentration10,11 devices using the nonlinear ICP.
Even with such high flexibility and robustness, it is still a practical challenge to clarify the operating conditions for the nonlinear ICP devices. The nonlinear regime of the ICP quickly removes cations through a cation exchange membrane, which causes the displacement of anions moving towards the anode. As a result, the flat depletion zone propagates quickly, which is reminiscent of shock propagation22. Mani et al. called this dynamic the deionization (or depletion) shock23. To preconcentrate targets at a designated sensing position, preventing the expansion of the ion depletion zone is necessary, for example, by applying EOF or pressure-driven flow against the zone expansion24. Zangle et al.22 clarified the criteria for ICP propagation in a one-dimensional model, and it highly depends on electrophoretic mobility17, ionic strength18, pH25, and so on. This indicates that proper operating conditions will be altered according to the sample conditions.
Here, we present detailed design and experimental protocols for a novel ICP platform that preconcentrates targets within a spatiotemporally defined position26. The expansion of the ion depletion zone is blocked by the ion enrichment zone, leaving a stationary preconcentration plug at an assigned position, regardless of the operating time, applied voltage, ionic strength, and pH. This detailed video protocol is intended to show the simplest method to integrate cation exchange membranes into microfluidic devices and to demonstrate the preconcentration performance of the new ICP platform compared to the conventional one.

<table border="0" cellpadding="0" cellspacing="0" width="720">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Sylgard 184 Silicone Elastomer kit</td>
			<td style="width:195px;">Dow Corning</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Trichlorosilane</td>
			<td style="width:195px;">Sigma Aldrich</td>
			<td style="width:119px;">175552</td>
			<td>Highly toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nafion perfluorinated resin, 20 wt%</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">527122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">71394</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium chloride</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">60121</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 carboxylic acid, succinimidyl ester</td>
			<td>Invitrogen</td>
			<td style="width:119px;">A20000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Isothiocyanate-conjugated albumin</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">A9771</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffer saline, 1X</td>
			<td>Wengene</td>
			<td style="width:119px;">LB004-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween 20&#160;</td>
			<td>Sigma Aldrich</td>
			<td style="width:119px;">P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epifluorescence microscope</td>
			<td>Olympus</td>
			<td style="width:119px;">IX-71</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Charged-coupled device camera</td>
			<td>Hamamtsu Co.</td>
			<td style="width:119px;">ImageEM X2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Source measurement unit</td>
			<td>Keithley Instruments</td>
			<td style="width:119px;">2635A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Covance-MP</td>
			<td style="width:195px;">Femto Science</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

merging ion concentration polarization between juxtaposed ion exchange membranes to block the propagation of the polarization zone

The ion concentration polarization (ICP) phenomenon is one of the most prevailing methods to preconcentrate low-abundance biological samples. The ICP induces a noninvasive region for charged biomolecules (i.e., the ion depletion zone), and targets can be preconcentrated on this region boundary. Despite the high preconcentration performances with ICP, it is difficult to find the operating conditions of non-propagating ion depletion zones. To overcome this narrow operating window, we recently developed a new platform for spatiotemporally fixed preconcentration. Unlike preceding methods that only use ion depletion, this platform also uses the opposite polarity of the ICP (i.e., ion enrichment) to stop the propagation of the ion depletion zone. By confronting the enrichment zone with the depletion zone, the two zones merge together and stop. In this paper, we describe a detailed experimental protocol to build this spatiotemporally defined ICP platform and characterize the preconcentration dynamics of the new platform by comparing them with those of the conventional device. Qualitative ion concentration profiles and current-time responses successfully capture the different dynamics between the merged ICP and the stand-alone ICP. In contrast to the conventional one that can fix the preconcentration location at only ~5 V, the new platform can produce a target-condensed plug at a specific location in the broad ranges of operating conditions: voltage (0.5-100 V), ionic strength (1-100 mM), and pH (3.7-10.3).

The schematic fabrication steps of a membrane-integrated microfluidic preconcentrator are shown in Figure 1. A detailed description of the fabrication is given in the Protocol. The designs and device images of the spatiotemporally defined preconcentrator26 are contrasted with those of a conventional preconcentrator11 (Figure 2). The ICP phenomenon in the spatiotemporally defined preconcentrator was investiga...

Watch this Scientific Journal Video about Merging Ion Concentration Polarization between Juxtaposed Ion Exchange Membranes to Block the Propagation of the Polarization Zone at JoVE.com

Merging Ion Concentration Polarization between Juxtaposed Ion Exchange Membranes to Block the Propagation of the Polarization Zone

The protocol for a novel ion concentration polarization (ICP) platform that can stop the propagation of the ICP zone, regardless of the operating conditions is described. This unique ability of the platform lies in the use of merging ion depletion and enrichment, which are two polarities of the ICP phenomenon.

The ion concentration polarization (ICP) phenomenon is one of the most prevailing methods to preconcentrate low-abundance biological samples. The ICP ...

The overall goal of this experiment is to preconcentrate bioagents by confining the ion concentration polarization zone between two identical ion exchange membranes. This method can help answer key questions in the field such as how to detect low abundance biomolecule when its concentration is less than the sensor's limit of detection. The main advantage of this technique is that we can generate ICP zone and preconcentrated bioagent in a very specific collision regardless of operation conditions.To begin this procedure, use conventional photolithography or deep reactive ion etching to fabricate silicon masters for the polydimethylsiloxane microchannel and cation-selective membrane molds. Place the silicon masters in a vacuum desiccator with about 30 microliters of trichlorosilane. Close the desiccator and salinize the silicon masters for 30 minutes.Next, mix together a silicone elastomer base and a curing agent in a 10 to one by weight ratio to obtain uncured PDMS. Degas the mixture under vacuum for 30 minutes. Then pour the degassed uncured PDMS onto the silicon masters.Remove bubbles from the PDMS with a handheld blower and then cure the PDMS molds at 80 degrees Celsius for two hours. Separate the cured PDMS components from the silicon masters. Use a knife to shape each component into a rectangle.Next, cut two lines in the cation-selective membrane mold from the edge of the mold to the prefabricated microchannels. Use a two millimeter biopsy punch to punch a hole at the end of each L-shaped channel. Clean a glass slide in the micromachine surface of the cation exchange membrane mold with acrylic adhesive tape and a blower.Place the clean face of the mold on the glass slide to reversibly seal the mold to the slide. Place 10 microliters of cation exchange resin on the glass slide in contact with the ends of the L-shaped channels. Place the tip of a syringe over the microchannel holes and gently withdraw the plunger to pull the resin into the channels.Within one minute of filling the resin channels, carefully detach the mold without touching the patterned resin on the slide. Heat the slide at 95 degrees Celsius for five minutes to evaporate the solvent from the resin. Use a razor blade to remove unneeded sections of cation exchange resin from the slide to form L-shaped cation-selective membranes.Next, use the biopsy punch to punch a 2.0 millimeter reservoir at each end of the prefabricated microchannel in the PDMS microchannel component. Then punch two more holes in the PDMS corresponding to the ends of the L-shaped membranes. Treat the PDMS microchannel component and the membrane-patterned substrate with oxygen plasma for 40 seconds at 100 watts and 50 Place the treated face of the substrate on the treated face of the PDMS microchannel ensuring that the cation-selective membranes cross the microchannel and are in lined with the holes in the PDMS.Apply gentle pressure as needed to seal the PDMS component to the substrate to complete device assembly. To begin the experiment, obtain several test ionic solutions with varying concentrations and pHs. Prepare a buffer solution of one molar potassium chloride or sodium chloride.Add a small amount of negatively charged fluorescent dye to each test solution ensuring that the dye concentration is low enough that its contribution to an electrical current solution is negligible. Load the first test solution into one reservoir of the microchannel. Draw the solution into the microchannel by applying gentle negative pressure to the other microchannel reservoir.Place a large droplet of the test solution over both microchannel reservoirs to eliminate any pressure gradient in the channel. Then fill the cation-selective membrane reservoirs with the chosen buffer solution to compensate for the ICP effect. Load the ICP chip onto an inverted epifluorescence microscope equipped with a charge-coupled device camera.Connect an anode to the left membrane reservoir and a cathode to the right membrane reservoir. Connect the electrodes to a source measure unit. Use the source measure unit to apply a voltage to the device and measure the current response.Acquire fluorescent images of the chip during voltage application. After the experiment, analyze the fluorescence intensity with the appropriate imaging software. Using this method, a microfluidic preconcentrator was fabricated and the current voltage time responses in fluorescence intensity profiles corresponding to various test solutions were investigated.As in conventional single membrane preconcentrators, three different voltage regimes were observed. During the Ohmic and limiting regimes, the linear concentration gradient developed at the cation-selective membranes and merged after about a second. Unlike single membrane preconcentrators, in the over-limiting regime, the ICP zones merged in less than a second with depletion shock observable by fluorescence imaging.The corresponding drop in conductivity was reflected in the current time response. The current time then recovered. This was observed at several voltages in the over-limiting regime.The current recovery was attributed to connective transport by ICP zones combined between the cation-selective membranes which isolate the preconcentration plugs throughout the voltage application. This current recovery was not observed in conventional ICP preconcentrators as the depletion zone and preconcentration plug freely propagate along the microchannel. While increased ionic strength, acidity and basicity decreased the relative intensity of preconcentration plugs in the spatiotemporally-defined preconcentrator, the plugs were still confined.Additional tests approaching preconcentration indicated that wider cation exchange membranes in a narrower channel can facilitate ICP preconcentration in these unfavorable conditions. Once mastered, this technique can be done in 30 minutes if it is performed properly. After its development, this technique paved the way for researchers in the field of electrokinetics to explore applications in microfluidic systems.After watching this video, you should have a good understanding of how to integrate ion exchange materials between microfluidic systems and how to use this platform to preconcentrate bioagents.

merging-ion-concentration-polarization-between-juxtaposed-ion

Research

JoVE Journal

Bioengineering

Fluorescent Nanoparticles for the Measurement of Ion Concentration in Biological Systems

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion fluxes at the cellular level are required for initiating action potentials in excitable cells.2 Sodium regulation plays an important role in both of these cases; however, no method currently exists for continuously monitoring sodium levels in vivo 3 and intracellular sodium probes 4 do not provide similar detailed results as calcium probes. In an effort to fill both of these voids, fluorescent nanosensors have been developed that can monitor sodium concentrations in vitro and in vivo.5,6 These sensors are based on ion-selective optode technology and consist of plasticized polymeric particles in which sodium specific recognition elements, pH-sensitive fluorophores, and additives are embedded.7-9 Mechanistically, the sodium recognition element extracts sodium into the sensor. 10 This extraction causes the pH-sensitive fluorophore to release a hydrogen ion to maintain charge neutrality within the sensor which causes a change in fluorescence. The sodium sensors are reversible and selective for sodium over potassium even at high intracellular concentrations.6 They are approximately 120 nm in diameter and are coated with polyethylene glycol to impart biocompatibility. Using microinjection techniques, the sensors can be delivered into the cytoplasm of cells where they have been shown to monitor the temporal and spatial sodium dynamics of beating cardiac myocytes.11 Additionally, they have also tracked real-time changes in sodium concentrations in vivo when injected subcutaneously into mice.3 Herein, we explain in detail and demonstrate the methodology for fabricating fluorescent sodium nanosensors and briefly demonstrate the biological applications our lab uses the nanosensors for: the microinjection of the sensors into cells; and the subcutaneous injection of the sensors into mice.

Fluorescent nanoparticles produced in our lab are used for imaging ion concentrations and ion fluxes in biological systems such as cells during signaling and interstitial fluid during physiological homeostasis.

Tightly regulated ion homeostasis throughout the body is necessary for the prevention of such debilitating states as dehydration.1 In contrast, rapid ion ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Multi-analyte Biochip (MAB) Based on All-solid-state Ion-selective Electrodes (ASSISE) for Physiological Research

Lab-on-a-chip (LOC) applications in environmental, biomedical, agricultural, biological, and spaceflight research require an ion-selective electrode (ISE) that can withstand prolonged storage in complex biological media 1-4. An all-solid-state ion-selective-electrode (ASSISE) is especially attractive for the aforementioned applications. The electrode should have the following favorable characteristics: easy construction, low maintenance, and (potential for) miniaturization, allowing for batch processing. A microfabricated ASSISE intended for quantifying H+, Ca2+, and CO32- ions was constructed. It consists of a noble-metal electrode layer (i.e. Pt), a transduction layer, and an ion-selective membrane (ISM) layer. The transduction layer functions to transduce the concentration-dependent chemical potential of the ion-selective membrane into a measurable electrical signal.
The lifetime of an ASSISE is found to depend on maintaining the potential at the conductive layer/membrane interface 5-7. To extend the ASSISE working lifetime and thereby maintain stable potentials at the interfacial layers, we utilized the conductive polymer (CP) poly(3,4-ethylenedioxythiophene) (PEDOT) 7-9 in place of silver/silver chloride (Ag/AgCl) as the transducer layer. We constructed the ASSISE in a lab-on-a-chip format, which we called the multi-analyte biochip (MAB) (Figure 1).
Calibrations in test solutions demonstrated that the MAB can monitor pH (operational range pH 4-9), CO32- (measured range 0.01 mM - 1 mM), and Ca2+ (log-linear range 0.01 mM to 1 mM). The MAB for pH provides a near-Nernstian slope response after almost one month storage in algal medium. The carbonate biochips show a potentiometric profile similar to that of a conventional ion-selective electrode. Physiological measurements were employed to monitor biological activity of the model system, the microalga Chlorella vulgaris.
The MAB conveys an advantage in size, versatility, and multiplexed analyte sensing capability, making it applicable to many confined monitoring situations, on Earth or in space.
Biochip Design and Experimental Methods
The biochip is 10 x 11 mm in dimension and has 9 ASSISEs designated as working electrodes (WEs) and 5 Ag/AgCl reference electrodes (REs). Each working electrode (WE) is 240 &mu;m in diameter and is equally spaced at 1.4 mm from the REs, which are 480 &mu;m in diameter. These electrodes are connected to electrical contact pads with a dimension of 0.5 mm x 0.5 mm. The schematic is shown in Figure 2.
Cyclic voltammetry (CV) and galvanostatic deposition methods are used to electropolymerize the PEDOT films using a Bioanalytical Systems Inc. (BASI) C3 cell stand (Figure 3). The counter-ion for the PEDOT film is tailored to suit the analyte ion of interest. A PEDOT with poly(styrenesulfonate) counter ion (PEDOT/PSS) is utilized for H+ and CO32-, while one with sulphate (added to the solution as CaSO4) is utilized for Ca2+. The electrochemical properties of the PEDOT-coated WE is analyzed using CVs in redox-active solution (i.e. 2 mM potassium ferricyanide (K3Fe(CN)6)). Based on the CV profile, Randles-Sevcik analysis was used to determine the effective surface area 10. Spin-coating at 1,500 rpm is used to cast ~2 &mu;m thick ion-selective membranes (ISMs) on the MAB working electrodes (WEs).
The MAB is contained in a microfluidic flow-cell chamber filled with a 150 &mu;l volume of algal medium; the contact pads are electrically connected to the BASI system (Figure 4). The photosynthetic activity of Chlorella vulgaris is monitored in ambient light and dark conditions.

Multi-analyte Biochip MAB Based on All-solid-state Ion-selective Electrodes ASSISE for Physiological Research

All-solid-state ion-selective electrodes (ASSISEs) constructed from a conductive polymer (CP) transducer provide several months of functional lifetime in liquid media. Here, we describe the fabrication and calibration process of ASSISEs in a lab-on-a-chip format. The ASSISE is demonstrated to have maintained a near-Nernstian slope profile after prolonged storage in complex biological media.

Lab-on-a-chip (LOC) applications in environmental, biomedical, agricultural, biological, and spaceflight research require an ion-selective electrode (ISE) ...

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after merging (FRAM). FRAM adopts the same principles as the fluorescence recovery after photobleaching (FRAP) technique, especially for measuring fluorescence recovery after bleaching a specific area, but FRAM uses a drop coalescence instead of photobleaching dye molecules to induce a chemical potential gradient of dye molecules. Our technique has several advantages over FRAP: it only requires a fluorescence microscope rather than a confocal microscope equipped with high power lasers; it is essentially free from the selection of fluorescence dyes; and it has far more freedom to define the measured diffusion area. Furthermore, FRAM potentially provides a route for studying the mixing or inter-diffusion of two different surfactants, when the monolayers at a surface of droplet and at a flat air/water interface are prepared with different species, independently.

We present a new technique to measure the lateral diffusion of a surface active species at the fluid-fluid interface by merging a droplet monolayer onto a flat monolayer.

We introduce a new method to measure the lateral diffusivity of a surfactant monolayer at the fluid-fluid interface, called fluorescence recovery after ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the cognitive powers of the brain with comparable efficiency and density. To date, silicon-based three-terminal transistors and two-terminal memristors have been widely used in neuromorphic circuits, in large part due to their ability to co-locate information processing and memory. Yet these devices cannot achieve the interconnectivity and complexity of the brain because they are power-hungry, fail to mimic key synaptic functionalities, and suffer from high noise and high switching voltages. To overcome these limitations, we have developed and characterized a biomolecular memristor that mimics the composition, structure, and switching characteristics of biological synapses. Here, we describe the process of assembling and characterizing biomolecular memristors consisting of a 5 nm-thick lipid bilayer formed between lipid-functionalized water droplets in oil and doped with voltage-activated alamethicin peptides. While similar assembly protocols have been used to investigate biophysical properties of droplet-supported lipid membranes and membrane-bound ion channels, this article focuses on key modifications of the droplet interface bilayer method essential for achieving consistent memristor performance. Specifically, we describe the liposome preparation process and the incorporation of alamethicin peptides in lipid bilayer membranes, and the appropriate concentrations of each constituent as well as their impact on the overall response of the memristors. We also detail the characterization process of biomolecular memristors, including measurement and analysis of memristive current-voltage relationships obtained via cyclic voltammetry, as well as short-term plasticity and learning in response to step-wise voltage pulse trains.

Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane.

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the ...

Focused Ion Beam Lithography to Etch Nano-architectures into Microelectrodes

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of sophisticated microelectrodes with greater resolution and expanded capabilities. The progress in fabrication technology has supported the development of biomimetic electrodes, which aim to seamlessly integrate into the brain parenchyma, reduce the neuroinflammatory response observed after electrode insertion and improve the quality and longevity of electrophysiological recordings. Here we describe a protocol to employ a biomimetic approach recently classified as nano-architecture. The use of focused ion beam lithography (FIB) was utilized in this protocol to etch specific nano-architecture features into the surface of non-functional and functional single shank intracortical microelectrodes. Etching nano-architectures into the electrode surface indicated possible improvements of biocompatibility and functionality of the implanted device. One of the benefits of using FIB is the ability to etch on manufactured devices, as opposed to during the fabrication of the device, facilitating boundless possibilities to modify numerous medical devices post-manufacturing. The protocol presented herein can be optimized for various material types, nano-architecture features, and types of devices. Augmenting the surface of implanted medical devices can improve the device performance and integration into the tissue.

We have shown that the etching of nano-architecture into intracortical microelectrode devices may reduce the inflammatory response and has the potential to improve electrophysiological recordings. The methods described herein outline an approach to etch nano-architectures into the surface of non-functional and functional single shank silicon intracortical microelectrodes.

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of ...

Propagation of Dental and Respiratory Cells and Organs in Microgravity

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) mimic the loss of gravity as it occurs in space and instead provide a microgravity environment through continuous rotation of cultured cells or tissues. These RCCSs ensure an un-interrupted supply of nutrients, growth and transcription factors, and oxygen, and address some of the shortcomings of gravitational forces in motionless 2D (two dimensional) cell or organ culture dishes. In the present study we have used RCCSs to co-culture cervical loop cells and dental pulp cells to become ameloblasts, to characterize periodontal progenitor/scaffold interactions, and to determine the effect of inflammation on lung alveoli. The RCCS environments facilitated growth of ameloblast-like cells, promoted periodontal progenitor proliferation in response to scaffold coatings, and allowed for an assessment of the effects of inflammatory changes on cultured lung alveoli. This manuscript summarizes the environmental conditions, materials, and steps along the way and highlights critical aspects and experimental details. In conclusion, RCCSs are innovative tools to master the culture and 3D (three dimensional) growth of cells in vitro and to allow for the study of cellular systems or interactions not amenable to classic 2D culture environments.

This protocol presents a method for the culture and 3D growth of ameloblast-like cells in microgravity to maintain their elongated and polarized shape as well as enamel-specific protein expression. Culture conditions for the culture of periodontal engineering constructs and lung organs in microgravity are also described.

Gravity is one of the key determinants of human cell function, proliferation, cytoskeletal architecture and orientation. Rotary bioreactor systems (RCCSs) ...

Employing Aeroponic Systems for the Clonal Propagation of Cannabis

This protocol describes the standardization of an efficient clonal propagation technique of hemp by utilizing aeroponic systems. Primary shoot cuttings were excised from two hemp varieties, named &#34;Cherry Wine&#34; and &#34;Red Robin&#34; (17-20% w/w CBD), that served as &#39;mother plant&#39;. An auxin precursor (indole-3-butyric acid) was applied to stimulate root development in the basal portion of the excised cuttings prior to placement in the system. Cuttings were lightly misted with the nutrient mist solution every three days to provide nutritional support as the solution contains the essential macronutrients, including nitrogen, phosphorus, and potassium. The aeroponic system water reservoir maintained a pH range between 5.0-6.0 and a water temperature between 20-22 &#176;C. A submersible water pump was used to deliver water to the cuttings. The shoot tip cuttings were provided with 24 h of light per day for 10 days until root development occurred, upon which the rooted cuttings were transplanted for research purposes. These aeroponic systems have proven to generate desirable results for Cannabis propagation. The method described here alleviates potential time constraints that arise from traditional methods to allow for a more efficient means for the asexual propagation of Cannabis.

This protocol is designed to provide instructional information for the clonal propagation of Cannabis sativa L. by implementing aeroponic systems. The method described here includes all necessary supplies and protocols to successfully reproduce desirable morphological and chemical properties in the genus Cannabis.

This protocol describes the standardization of an efficient clonal propagation technique of hemp by utilizing aeroponic systems. Primary shoot cuttings ...