Name: assembly and characterization of biomolecular memristors consisting of ion channel-doped lipid membranes
Uploaded: 2019-03-09T15:00:00
Description: Watch this Scientific Journal Video about Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes at JoVE.com

Financial support was provided by the National Science Foundation Grant NSF ECCS-1631472. Research for G.J.T., C.D.S., A.B., and C.P.C. was partially sponsored by the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory, managed by UT-Battelle, LLC, for the U.S. Department of Energy. A portion of this research was conducted at the Center for Nanophase Materials Sciences, which is a DOE Office of Science User Facility.&#160;

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This paper presents a protocol for assembling and characterizing biomolecular memristors based on ion channel-doped synthetic biomembranes formed between two droplets of water in oil. The soft-matter, two-terminal device is designed and studied to: 1) overcome constraints that are associated with solid-state technology, such as high noise, high energy consumption, and high switching voltages, 2) more closely mimic the composition, structure, switching mechanisms of biological synapses, and 3) explore the mechanisms and f...

It is widely recognized that biological synapses are responsible for the high efficiency and enormous parallelism of the brain due to their ability to learn and process information in highly adaptive ways. This coordinated functionality emerges from multiple, highly complex molecular mechanisms that drive both short-term and long-term synaptic plasticity1,2,3,4,5. Neuromorphic computing systems aim to emulate synaptic functionalities at levels approaching the density, complexity, and energy efficiency of the brain, which are needed for the next generation of brain-like computers6,7,8. However, reproducing synaptic features using traditional electronic circuit elements is virtually impossible9, instead requiring the design and fabrication of new hardware elements that can adapt to incoming signals and remember information histories9. These types of synapse-inspired hardware are known as mem-elements9,10,11 (short for memory elements), which, according to Di Ventra et al.9,11, are passive, two-terminal devices whose resistance, capacitance, or inductance can be reconfigured in response to external stimuli, and which can remember prior states11. To achieve energy consumption levels approaching those in the brain, these elements should employ similar materials and mechanisms for synaptic plasticity12.
To date, two-terminal memristors13,14,15 have predominantly been built using complementary metal-oxide-semiconductor (CMOS) technology, characterized by high-switching voltages and high noise. This technology does not scale well due to high power consumption and low density. To address these limitations, multiple organic and polymeric memristors have been recently built. However, these devices exhibit significantly slower switching dynamics due to time-consuming ion diffusion through a conductive polymer matrix16,17. As a result, the mechanisms by which both CMOS-based and organic memristive devices emulate synapse-inspired functionalities are highly phenomenological, encompassing only a few synaptic functionalities such as Spike Timing Dependent Plasticity (STDP)18, while overlooking other key features that also play essential roles in making the brain a powerful and efficient computer, such as pre-synaptic, short-term plasticity19.&#160;
Recently, we introduced a new class of memristive devices12 featuring voltage-activated peptides incorporated in biomimetic lipid membranes that mimics the biomolecular composition, membrane structure, and ion channel triggered switching mechanisms of biological synapses20.&#160; Here, we describe how to assemble and electrically interrogate these two-terminal devices, with specific focus on how to evaluate short-term plasticity for implementation in online learning applications12. Device assembly is based on the droplet interface bilayer (DIB)21 method, which has been used extensively in recent years to study the biophysics of model membranes21 and membrane-bound ion channels22,23,24, and as building blocks for the development of stimuli-responsive materials25,26. We describe the membrane assembly and interrogation process in detail for those interested in neuromorphic applications but have limited experience in biomaterials or membrane biology. The protocol also includes a full description of the characterization procedure, which is as important as the assembly process, given the dynamic and reconfigurable electrical properties of the device27. The procedure and representative results described here are foundations for a new class of low-cost, low-power, soft mem-elements based on lipid interfaces and other biomolecules for applications in neuromorphic computing, autonomous structures and systems, and even adaptive brain-computer interfaces.

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			<td style="width:183px;">850356P/850356C</td>
			<td style="width:663px;">Purchased as lyophilized powder (P) or in chloroform (C)&#160;</td>
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			<td style="width:183px;">610000</td>
			<td>This includes a mini-extruder, 2 syringes, 100 PC membranes, 100 filter supports, and 1 holder/heating block</td>
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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.

Figure 1 displays the experimental setup used to assemble and characterize the biomolecular memristor. Lowering the free ends of the electrodes to the bottom of the oil reservoir, as shown in Figure 1b, was found helpful to minimize vibrations of the electrodes and droplets that can result in variations in measured current and bilayer area, especially in cases where heating the oil can generate convective flow in the oil. Figure 2 s...

Watch this Scientific Journal Video about Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes at JoVE.com

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

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 ...

Our protocol describes how to assemble and electrically characterize a peptide-doped biomembrane that closely mimics the composition, structure, and transport properties of biological synapses and which exhibits tunable memory resistance. This technique empowers users to assess activity-dependent, memory resistance and short-term plasticity in engineered systems at time scales and excitation levels relevant to biological synapses and ion channels. This technique provides a framework for characterizing biomimetic membranes containing voltage-activated ion channels, making it applicable to characterization of a variety of cellular transport processes, including those in neurons.Our suggestion to new researchers is to first become proficient at preparing liposome solutions and assembling a droplet interface bilayer on wire-type electrodes. Seeing firsthand the process for droplet dispensing and positioning on electrodes simplifies this technique for bilayer formation, making it immediately accessible to all. Demonstrating this procedure will be Dr.Joseph Najem, a postdoc from my laboratory.To begin, prepare the alamethicin stock solution in the microcentrifuge tube by dissolving the alamethicin peptides powder in ethanol to a final concentration of 2.5 milligram per milliliter. Vortex the tube briefly to mix well, and store the stock solution in a minus 20 degree Celsius freezer. In a 1.5 milliliter Safe-Lock tube, add one microliter of alamethicin stock solution to 99 microliters of solution A to achieve a final alamethicin concentration of 13 micromolar in the liposome suspension.Vortex to mix well. The resulting peptide liposome solution is solution B.Mix 117 microliters of solution A with 10 microliters of solution B to achieve a final alamethicin concentration of one micromolar, and then vortex to mix well. Refer to the resulting solution as C.Store the solutions B and C at four degrees Celsius.Place a one-millimeter thick 25 times 75 millimeters glass side on the stage of an inverted microscope. Dispense a few drops of hexadecane oil onto the center of the glass slide, and then place the oil reservoir directly onto the oil on the glass slide. Completely fill the oil reservoir with hexadecane oil.Make sure the reservoir is positioned above the objective lens. Then plug the electrode holder to the head stage of a current amplifier mounted on a micromanipulator. The micromanipulator minimizes electrode length and electrical noise.Then, mount the glass micropipette holder with the second silver-silver-chloride wire onto another micromanipulator. Using the micromanipulators, position the electrodes such that the agarose-coated tips of the silver-silver-chloride wires are fully submerged into the oil reservoir at a similar vertical plane. Align the two electrodes and separate them by a few millimeters.To form the lipid bilayer, move the electrodes vertically into the oil phase. Use the micropipette to deposit 200 nanoliters of lipid solution A on each of the wires. Wait for three to five minutes to allow for spontaneous lipid monolayer assembly to occur at the water oil interface.Droplets might sag if the surrounding oil is sufficiently less dense. After that, lower the electrodes to re-submerge until the ends of both electrodes barely touch the bottom of the oil reservoir. Then to form the bilayer, move the electrodes horizontally to bring the droplets into contact.To obtain the pinched, hysteretic, current-voltage relationship, use a function generator to apply triangular or sinusoidal voltage waveform to an alamethicin-free lipid membrane assembled with droplets of solution A.Record the induced current response across multiple frequencies. To record the size of the interfacial lipid bilayer, either measure the diameter of the lipid membrane on computer or record the peak-to-peak current amplitude resulting from the 10 hertz, 10 millivolts triangular wave to calculate the area of the membrane. Take the wires out of the oil phase to remove the droplets that contain no alamethicin.Add new aqueous droplets using solution C and form a lipid bilayer. Based on the square wave current amplitude, use the micromanipulators to adjust contact between droplets, such that the bilayer has a similar area as the one formed earlier. Then apply a 10-hertz and 10-millivolts voltage wave form, and record the induced current response as previously.To conduct pulse experiments using a custom programming software and analog voltage source, generate voltage pulses with specific high and low amplitudes on time and off time. Record the current in response to applied pulses. The plot of current versus voltage shows the non-zero current response upon application of a voltage bias to an alamethicin-free lipid bilayer.Add 0.017 hertz, a frequency where the impedance is dominated by membrane resistance. A low ohmic current response is shown for the highly insulating membrane. The plot of a lipid bilayer formed between two droplets containing alamethicin peptides shows exponentially increasing currents at voltages higher than the insertion threshold of 100 millivolts.At high voltage, alamethicin peptides residing at the surface of the lipid bilayer insert into the membrane and aggregate to form conductive pores. The symmetric current responses at both polarities is due to the insertion and aggregation of separate populations of peptides from opposite sides of the membrane. The capacitive current must be subtracted from the total current to obtain only the memristive, pinched hysteresis current-voltage response.The biomolecular memristor response to subsequent voltage pulses with an increase in conductance during the on time, despite intermittently restoring an insulating state during each off time. Both the present stimulus and prior stimuli contribute to the current increase. Cohesive monolayers on both droplets must form before bringing them together to form the bilayer.If the droplets are brought together too early, they coalesce and no bilayer is formed. We are now designing and fabricating, microfluidic base neural networks consisting of solid state neurons connected by membrane-based synapses supported by network assimilation optimization of high-performance supercomputers at ORNL. These memristors are the first to have the composition, structure, switching mechanism, and ion transport of biological synapses.They thus provide a biomolecular basis furnished adding brain-like computing and memory.

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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, ...

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation -&#160;is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.

In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.

In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.

The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.

Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

Isolation and Characterization Of Chimeric Human Fc-expressing Proteins Using Protein A Membrane Adsorbers And A Streamlined Workflow

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using affinity chromatography.&#160;While affinity chromatography using porous protein A agarose beads packed in columns is arguably the most common method of laboratory scale isolation of antibodies and recombinant proteins expressing Fc fragments of IgG, it can be a time consuming and expensive process.&#160;Time and financial constraints are especially daunting in small basic science labs that must recover hundreds of micrograms to milligram quantities of protein from dilute solutions, yet lack access to high pressure liquid delivery systems and/or personnel with expertise in bioseparations. Moreover, product quantification and characterization may also excessively lengthen processing time over several workdays and inflate expenses (consumables, wages, etc.).&#160;Therefore, a fast, inexpensive, yet effective protocol is needed for laboratory scale isolation and characterization of antibodies and other proteins possessing an Fc fragment.&#160;To this end, we have devised a protocol that can be completed by limited-experience technical staff in less than 9 hr (roughly one workday) and as quickly as 4 hr, as opposed to traditional methods that demand 20+ work hours. Most required equipment is readily available in standard biomedical science, biochemistry, and (bio)chemical engineering labs, and all reagents are commercially available.&#160;To demonstrate this protocol, representative results are presented in which chimeric&#160;murine galectin-1 fused to human Fc (Gal-1hFc) from cell culture supernatant was isolated using a protein A membrane adsorber.&#160;Purified Gal-1hFc was quantified using an expedited Western blotting analysis procedure and characterized using flow cytometry. The streamlined workflow can be modified for other Fc-expressing proteins, such as antibodies, and/or altered to incorporate alternative quantification and characterization methods.

Compared with traditional affinity chromatography using protein A agarose bead-packed columns, protein A membrane adsorbers can significantly speed laboratory-scale isolation of antibodies and other Fc fragment-expressing proteins.&#160;Appropriate analysis and quantification methods can further accelerate protein processing, allowing isolation/characterization to be completed in one workday, instead of 20+ work hours.

Laboratory scale to industrial scale purification of biomolecules from cell culture supernatants and lysed cell solutions can be accomplished using ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

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 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 ...

A Method for Growing Bio-memristors from Slime Mold

Our research is aimed at gaining a better understanding of the electronic properties of organisms in order to engineer novel bioelectronic systems and computing architectures based on biology. This specific paper focuses on harnessing the unicellular slime mold Physarum polycephalum to develop bio-memristors (or biological memristors) and bio-computing devices. The memristor is a resistor that possesses memory. It is the 4th fundamental passive&#160;circuit element (the other three are the resistor, the capacitor, and the inductor), which is paving the way for the design of new kinds of computing systems; e.g., computers that might relinquish the distinction between storage and a central processing unit. When applied with an AC voltage, the current vs. voltage characteristic of a memristor is a pinched hysteresis loop. It has been shown that P. polycephalum produces pinched hysteresis loops under AC voltages and displays adaptive behavior that is comparable with the functioning of a memristor. This paper presents the method that we developed for implementing bio-memristors with P. polycephalum and introduces the development of a receptacle to culture the organism, which facilitates its deployment as an electronic circuit component. Our method has proven to decrease growth time, increase component lifespan, and standardize electrical observations.

This paper introduces an improved method for growing bio-memristors out of the plasmodium of Physarum polycephalum. Such a method has proven to decrease growth time, increase component lifespan, standardize electrical observations, and create a protected environment that can be integrated into conventional circuitry.

Our research is aimed at gaining a better understanding of the electronic properties of organisms in order to engineer novel bioelectronic systems and ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

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 ...