This work was supported by the Australian Research Council (ARC) Discovery Program (DP150101065) and the ARC Research Hub for Integrated Device for End-user Analysis at Low-levels (IDEAL) (IH150100028).

1.&#160;tBLMs electrodes preparation
<ol>
	<li>Preparation of first monolayer coating
	<ol>
		<li>Immerse a freshly sputtered gold patterned electrode microscope slide in an ethanolic solution comprised of a 3 mM 1:9 ratio of benzyl-disulfide-tetra-ethyleneglycol-OH &#34;spacer&#34; molecules (benzyl disulfide comprised a four oxygen-ethylene glycol spacer, terminated with an OH group) and benzyl-disulfide (tetra-ethyleneglycol) n=2 C20-phytanyl &#34;tethered&#34; molecules. This creates the first layer coating to which a bilayer can be anchored. 
		&#8203;NOTE: The gold electrode is made by evaporating 100 nm, 99.9995% gold (5n5 gold) film onto custom 25 mm x 75 mm polycarbonate slides20.</li>
		<li>Incubate electrodes with the first layer at room temperature for at least 1 h.</li>
		<li>Rinse the gold electrodes by immersing in copious amounts of pure ethanol over 30 s.</li>
		<li>Use the gold electrode slide with the first monolayer directly for the next step or store in a jar full of pure ethanol.</li>
		<li>NOTE: To ensure the integrity of the first layer, minimize any direct contact to the gold portions of the slide</li>
	</ol>
	</li>
	<li>Assembling the first monolayer coated slide
	<ol>
		<li>Carefully take off one coplanar gold electrode slide from its container using tweezers, being sure not to make contact with the patterned areas where the tBLMs will form. 
		NOTE: Be mindful to identify the side of the slide onto which the gold is deposited.</li>
		<li>Air dry slide for 1 - 2 min in to remove any residual ethanol.</li>
		<li>Place gold electrode over a dry surface, ensure the gold electrode is correctly oriented with patterned gold surface facing up.</li>
		<li>Peel the transparent adhesive layer cover from a thin laminate and place over the 6 channels to define each well.</li>
		<li>Use a pressure roller to release any air between the slide and transparent adhesive layer, as shown in&#160;Figure 1A. 
		NOTE: The time required for this step will need to be optimized by the researcher. In this protocol, times are ranging from 2-3 min.</li>
		<li>Introduce as soon as practicable (within 1-2 minutes) the second lipid bilayer to the assembled first monolayer coated electrode for self-assembly to avoid damaging the first layer.</li>
	</ol>
	</li>
	<li>Preparation of second lipid bilayer
	<ol>
		<li>Add 6 &#181;L of 3 mM lipids of interest to the first well of the six wells slide. Do not let the edge of the micropipette tip touch the gold surface, which can damage the tethered chemistries on the electrode. 
		NOTE: The lipid mixture used in this work consisted of 3 mM 70% zwitterionic C20 diphytanyl-ether-glycero-phosphatidylcholine (DPEPC) and 30% C20 diphytanyldiglyceride ether lipids (GDPE) mixed with 3 mM cholesterol-PEG-Biotin in 50:1 molar ratio.</li>
		<li>Introduce 6 &#181;L of the lipid mixture to the other wells with a 10 s gap between each addition.</li>
		<li>Incubate each well for exactly 2 min at room temperature before exchanging the lipid mixture over the electrodes with a buffer such as PBS. Space the times for the addition and buffer exchange 10 s apart so each well is incubated with the lipid for exactly 2 min each.</li>
		<li>Wash 3 more times with 50 &#181;L of PBS buffer (pH 7.0). Be sure to leave 50 &#181;L of buffer over the electrodes at all times. Do not allow the electrodes to dry. 
		NOTE: Displacing the ethanol solvent with the aqueous solution in this way (the&#160;solvent exchange method) enables the rapid formation of a single lipid bilayer anchored to the gold electrode via the tethered chemistries.</li>
	</ol>
	</li>
	<li>Testing tBLM formation using electrical impedance spectroscopy (EIS) measurements
	<ol>
		<li>Insert prepared electrode slide into an AC impedance spectrometer (e.g., Tethapod). Ensure that the spectrometer is connected via a USB port to a computer running the software.</li>
		<li>Open the software, click&#160;Setup&#160;and open&#160;Hardware.</li>
		<li>Set the hardware settings to use 25 mV peak-to-peak AC excitation.</li>
		<li>Set frequencies between 0.1 and 10,000 Hz with two steps per decade for rapid impedance measures press ok.</li>
		<li>Click the&#160;Setup&#160;menu and open&#160;Model.</li>
		<li>Use an equivalent circuit model that describes the tethering gold electrode as a constant phase element in series with a resistor describing the electrolyte buffer and a parallel resistor-capacitor network to describe the lipid bilayer, and press&#160;OK.</li>
		<li>Press the&#160;Start&#160;button in order to start a real-time measurement of membrane capacitance (Cm) and membrane conduction (Gm). Cm&#160;values of typical tBLMs should be in the range of 12.5 nF to 15.5 nF for 10% tethered chemistries21,22.</li>
		<li>After running the protocol and finishing the experiment, save the data.</li>
		<li>Repeat the measurement with the next well.</li>
	</ol>
	</li>
</ol>
2.&#160;Laser irradiation
<ol>
	<li>Experimental setup 
	NOTE: The custom-made system is set up for each tBLM well individually.
	<ol>
		<li>Perform experiments in a light-proof box to minimize laser hazardously.</li>
		<li>Use an optics table to set up the experiment to reduce unwanted vibrations.</li>
		<li>Place the impedance reader, where is the gold slide is connected, on an XYZ stage and elevate such that it sits in the path of the laser source.</li>
		<li>Use coarse-fine focusing microscopic gearing to control the height of the laser source to achieve the appropriate precision.</li>
		<li>Target the laser path along the longitudinal axis of the electrode slide. 
		CAUTION: Always wear suitable laser safety glasses and maintain good laser safety protocols.</li>
		<li>Allow the selected tuned laser to stabilize before starting the experiment. 
		NOTE: A schematic of the experimental setup is illustrated in&#160;Figure 2A.</li>
	</ol>
	</li>
	<li>Alignment of laser and gold electrodes 
	NOTE: Before beginning, always assess the laser power output using a power-meter to ensure only very low wattages are delivered to the tBLMs.
	<ol>
		<li>Adjust either the laser path or the angle of the electrode such that the laser passes through the liquid covering the electrode and is just visible, evenly, at the gold surface.</li>
		<li>Adjust the laser beam light position for each experiment by raising or lowering the laser beam source using the fine adjustment while observing changes in membrane conductance.</li>
		<li>Lock the knob to secure the position of the laser path when there are no conductance changes are observed. 
		NOTE: Increased membrane conductance values will be generated when the laser interacts with the underlying gold electrode. It is, therefore, important to adjust the laser path such that no such interactions are possible.</li>
	</ol>
	</li>
	<li>Sample preparation
	<ol>
		<li>Prepare the laser beam light alignment (where there is no change in membrane conductance), as shown in&#160;Figure 2, position 3.</li>
		<li>Add GNPs of interest (functionalized or bare) to the PBS buffer in which the tBLMs are immersed while the laser is switched OFF.</li>
		<li>Mix the PBS buffer surrounding the tBLMs gently three times, being careful not to touch the electrode.</li>
		<li>Incubate for 5-10 min at room temperature.</li>
		<li>Turn the laser ON to irradiate sample, using the correct aligned laser beam light position as seen in&#160;Figure 2, position 3.</li>
		<li>Use the appropriate combination of GNPs size, shape and concentration with laser light wavelength. 
		NOTE: The laser beam of set wavelength should couple to the corresponding GNP plasmon resonance frequency.</li>
		<li>Record measured current continuously (real-time measurements).</li>
		<li>Perform steps 2.2.1 - 2.3.7, omitting GNP addition for the control experiments.</li>
	</ol>
	</li>
</ol>
3.&#160;Statistical data analysis and presentation
<ol>
	<li>Export the data into a spreadsheet.</li>
	<li>Extract the membrane conductance parameter versus time.</li>
	<li>Use the recorded data after setting a laser beam light with the right position and prior to GNPs introduction.</li>
	<li>Normalize data by dividing the measured membrane conductance over the baseline membrane conductance. 
	NOTE: This confirms that relative changes in membrane conduction values elicited by introduced irradiated GNPs.</li>
	<li>Present data as plots of time (x-axis) versus normalized membrane conduction (y-axis).</li>
</ol>
4.&#160;Predict the amount of localized heat generated in the tBLMs from irradiated nanoparticles (thermal predictive model)
<ol>
	<li>Solve the radiation transfer problem according to Dombrovsky23, in order to calculate absorbed radiation power in irradiated nanoparticle solutions.</li>
	<li>Calculate the heat generation by incorporating the heat source due to absorbed radiation into the energy equation. 
	NOTE: For a detailed explanation of the numerical analysis of heat generation in the tBLMs from irradiated nanoparticles and the nanomaterial-electrode interface, refer to&#160;19.</li>
</ol>

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The authors declare the following financial interests/personal relationships, which may be considered as potential competing interests: Prof Bruce Cornell is Director - Science and Technology at Surgical Diagnostics SDx tethered membranes Pty. Ltd.

This protocol describes the use of tBLM model with a coplanar electrode substrate in conjunction with a horizontal laser alignment set up that enables the real-time electrical impedance recording in response to laser irradiation of gold nanoparticles. The method of EIS recording presented here constructs a minimal list of experiments necessary to provide recording of ion current changes across the membrane, which corresponds to the heat generated by the coupled laser and gold nanoparticle...

The hyperthermic performance of irradiated gold nanomaterials offers a new class of minimally invasive, selective, targeted treatment for infections and tumors1. The employment of nanoparticles that can be heated by a laser has been used to selectively destroy diseased cells as well as providing a means for selective drug delivery2,3. A consequence of the photothermolysis phenomena of heated plasmonic nanoparticles is damage to the cell membranes. The fluid lipid bilayer membrane is considered a particularly vulnerable site for cells undergoing such treatments because denaturation of intrinsic membrane proteins as well as membrane damage can also lead to cell death4, as many proteins are there to maintain the ionic potential gradient across cell membranes. While the ability to determine and monitor heat transfer at the nanoscale is of key interest to the study and application of irradiated GNPs1,5,6,7, assessment and understanding of the molecular interactions between GNPs and bio-membranes, as well as the direct consequences of the laser-induced heating phenomena of embedded GNPs in biological tissues, are yet to be fully elucidated8. Therefore, a thorough understanding of the hyperthermia process of irradiated GNPs remains a challenge. As such, the development of a nanomaterial-electrode interface that mimics the natural surroundings of cells could provide a means by which to undertake an in-depth investigation of the heat transfer characteristics of irradiated gold nanoparticles within biological systems.
The complexity of native cell membranes is one of the significant challenges in understanding the irradiated GNPs interactions in cells. There have been various artificial membrane platforms developed to provide close simple bio-mimetic versions of natural lipid membrane architecture and functionality, including, but not limited to, black lipid membranes9, supported planar bilayer membranes10, hybrid bilayer membranes11, polymer-cushioned lipid bilayer membranes12&#160;and tethered bilayer lipid membranes13. Each artificial lipid membrane model has distinct advantages and limitations with respect to mimicking the natural lipid membranes14.
This study describes the employment of lipid membrane-coated electrodes as a sensor for assessing gold nanoparticle and lipid membrane interactions, using the tBLM model. The tBLM based biosensor detection scheme provides inherent stability and sensitivity13&#160;as tethered membranes can self-repair, unlike other systems (such as membranes formed by patch-clamp or liposomes) in which only a small amount of membrane damage results in their collapse15,16,17,18. Further, because tBLMs are of mm2&#160;dimensions, the background impedance is orders of magnitude lower than patch-clamp recording techniques, which enables a recording of changes in basal membrane ionic flux due to nanoparticle interactions. As a result of this, the present protocol can contrast changes in membrane conductance by bound GNPs that are excited by lasers whose powers are as low as 135 nW/&#181;m2.
The system presented here provides a sensitive and reproducible method for determining precise laser parameters, particle size, particle coatings and composition needed to design and develop thermal therapies. This is critical for the refinement of emerging photothermal therapies, as well as offering valuable information for detailed mechanisms of heat transfer within biological systems. The presented protocol is based on previously published work19. An outline of the protocol is as follows: the first section defines the tBLM formation; the second section outlines how to construct the setup and align the excitation laser source; the final section illustrates how to extract information from the electrical impedance spectroscopy data.

<table><tbody><tr><td>30 nm diameter streptavidin-conjugated gold nanoparticles</td><td>Cytodiagnostics</td><td>AC-30-04-05</td><td>This is a streptavidin-conjugated GNPs product ready for use</td></tr><tr><td>30 nm diameter bare gold nanoparticles</td><td>Sigma-Aldrich</td><td>753629</td><td>This is a bare GNPs product ready for use</td></tr><tr><td>Cholesterol-PEG-Biotin (MW1000)</td><td>NANOCS</td><td>PG2-BNCS-10k</td><td>Dissolved in highly pure ethanol</td></tr><tr><td>C20 Diphytanyl-Glycero-Phosphatidylcholine lipids</td><td>SDx Tethered Membranes Pty. Ltd.</td><td>SDx-S1</td><td>1 ml glass vial containing 70% C16 diphytanyl phosphatidylcholine (DPEPC) and 30% C16 diphytanyl glycerol (GDPE) in 99.9% ethanol</td></tr><tr><td>Benzyl-disulfide-tetra-ethyleneglycol-OH</td><td>SDx Tethered Membranes Pty. Ltd.</td><td>SDx-S2</td><td>Spacer molecules</td></tr><tr><td>Benzyl-disulfide (tetra-ethyleneglycol) n=2 C20-phytanyl&amp;nbsp;</td><td>SDx Tethered Membranes Pty. Ltd.</td><td>SDx-S2</td><td>Tethered molecules</td></tr><tr><td>532 nm green laser continuous light</td><td>OBIS LS/OBIS CORE LS, China</td><td>ND-1000</td><td>The power of this laser was ~135&amp;nbsp;mW&amp;nbsp;</td></tr><tr><td>tethaPod EIS reader</td><td>SDx Tethered Membranes Pty. Ltd.</td><td>SDx-R1</td><td>A reader of conductance and capacitance on six channels simultaneously</td></tr><tr><td>tethaPlate cartridge assembly</td><td>SDx Tethered Membranes Pty. Ltd.</td><td>SDx-BG</td><td>Materials to attach the slide with electrodes to the flow cell cartridge</td></tr><tr><td>Clamp and slide assembly jig</td><td>SDx Tethered Membranes Pty. Ltd.</td><td>SDx-A1</td><td>Materials to attach the slide with electrodes to the flow cell cartridge</td></tr><tr><td>Lipid coated coplanar gold electrodes</td><td>SDx Tethered Membranes Pty. Ltd.</td><td>SDx-T10</td><td>Coplanar&amp;nbsp; gold electrodes are made from 25 mm x 75&amp;nbsp;mm x&amp;nbsp;1&amp;nbsp;mm polycarbonate base substrate with patterned gold arrays layout, then coated with benzyldisulphide, bis-tetraethylene glycol C16 phytanyl half membrane spanning tethers in a tether ratio of 10%&amp;nbsp;</td></tr><tr><td>tethaQuick software</td><td>SDx Tethered Membranes Pty. Ltd.</td><td>SDx-B1</td><td>Software for use with tethaPod to process data and display conductance, impedance and capacitance measurements from the tethaPlate electrodes</td></tr><tr><td>&amp;nbsp;99.9% Pure ethanol</td><td>Sigma-Aldrich&amp;nbsp;</td><td>34963</td><td>Absolute,&amp;nbsp; 99.9%</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich</td><td>P4417</td><td>pH 7</td></tr></tbody></table>

tethered bilayer lipid membranes to monitor heat transfer between gold nanoparticles and lipid membranes

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry using tethered bilayer lipid membranes (tBLMs) assembled on gold electrodes. Irradiated modified GNPs, such as streptavidin-conjugated GNPs, are embedded in tBLMs containing target molecules, such as biotin. By using this approach, the heat transfer processes between irradiated GNPs and model bilayer lipid membrane with entities of interest are mediated by a horizontally focused laser beam. The thermal predictive computational model is used to confirm the electrochemically induced conductance changes in the tBLMs. Under the specific conditions used, detecting heat pulses required specific attachment of the gold nanoparticles to the membrane surface, while unbound gold nanoparticles failed to elicit a measurable response. This technique serves as a powerful detection biosensor which can be directly utilized for the design and development of strategies for thermal therapies that permits optimization of the laser parameters, particle size, particle coatings and composition.

The gold substrate upon which tBLMs can be created is shown in&#160;Figure 1. A schematic of the experimental setup is presented in&#160;Figure 2.
Coplanar gold electrodes, as shown in&#160;Figure 1A, are made from 25 mm x 75 mm x 1 mm polycarbonate base substrate with patterned gold arrays. A transparent adhesive layer defines the six individual measuring chambers. The coplanar gold electrode allows the ...

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Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

This work outlines a protocol to achieve dynamic, non-invasive monitoring of heat transfer from laser-irradiated gold nanoparticles to tBLMs. The system combines impedance spectroscopy for the real-time measurement of conductance changes across the tBLMs, with a horizontally focused laser beam that drives gold nanoparticle illumination, for heat production.

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...

tethered-bilayer-lipid-membranes-to-monitor-heat-transfer-between

Research

JoVE Journal

Biology

2.6K Views.  University of Technology Sydney. Dit werk schetst een protocol om dynamische, niet-invasieve monitoring van warmteoverdracht van laserbestraalde gouden nanodeeltjes naar tBLMs te bereiken. Het systeem combineert impedantiespectroscopie voor de real-time meting van geleidingsveranderingen over de tBLMs, met een horizontaal gerichte laserstraal die gouden nanodeeltjesverlichting aandrijft, voor warmteproductie.

Video: Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

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 ...

Bioengineering

Monitoring Protein Adsorption with Solid-state Nanopores

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids 1-6, the unfolding of proteins 7, and binding affinity of different ligands 8. By coupling these nanopores to the resistive-pulse technique 9-12, such measurements can be done under a wide variety of conditions and without the need for labeling 3. In the resistive-pulse technique, an ionic salt solution is introduced on both sides of the nanopore. Therefore, ions are driven from one side of the chamber to the other by an applied transmembrane potential, resulting in a steady current. The partitioning of an analyte into the nanopore causes a well-defined deflection in this current, which can be analyzed to extract single-molecule information. Using this technique, the adsorption of single proteins to the nanopore walls can be monitored under a wide range of conditions 13. Protein adsorption is growing in importance, because as microfluidic devices shrink in size, the interaction of these systems with single proteins becomes a concern. This protocol describes a rapid assay for protein binding to nitride films, which can readily be extended to other thin films amenable to nanopore drilling, or to functionalized nitride surfaces. A variety of proteins may be explored under a wide range of solutions and denaturing conditions. Additionally, this protocol may be used to explore more basic problems using nanopore spectroscopy.

A method of using solid-state nanopores to monitor the non-specific adsorption of proteins onto an inorganic surface is described. The method employs the resistive-pulse principle, allowing for the adsorption to be probed in real-time and at the single-molecule level. Because the process of single protein adsorption is far from equilibrium, we propose the employment of parallel arrays of synthetic nanopores, enabling for the quantitative determination of the apparent first-order reaction rate constant of protein adsorption as well as and the Langmuir adsorption constant.

Solid-state nanopores have been used to perform measurements at the single-molecule level to examine the local structure and flexibility of nucleic acids ...

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

Biochemistry

Visualizing Diffusional Dynamics of Gold Nanorods on Cell Membrane using Single Nanoparticle Darkfield Microscopy

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and provides a theoretical basis for the rational design of nano-medicine delivery. Single particle tracking (SPT) analysis could probe the position and orientation of individual nanoparticles on cell membrane, and reveal their translational and rotational states. Here, we show how to use traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on live cell membrane. We also show how to extract the location and orientation of AuNRs using ImageJ and MATLAB, and how to characterize the diffusive states of AuNRs. Statistical analysis of hundreds of particles show that single AuNRs perform Brownian motion on the surface of U87 MG cell membrane. However, individual long trajectory analysis shows that AuNRs have two distinctly different types of motion states on the membrane, namely long-range transport and limited-area confinement. Our SPT methods can be potentially used to study the surface or intracellular particle diffusion in different biological cells and can become a powerful tool for investigations of complex cellular mechanisms.

Here, we show the use of traditional dark-field microscopy to monitor the dynamics of gold nanorods (AuNRs) on cell membrane. The location and orientation of single AuNRs are detected using ImageJ and MATLAB, and the diffusive states of AuNRs are characterized by single particle tracking analysis.

Analyzing the diffusional dynamics of nanoparticles on cell membrane plays a significant role in better understanding the cellular uptake process and ...

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

Nanoparticle Tracking Analysis of Gold Nanoparticles in Aqueous Media through an Inter-Laboratory Comparison

In the field of nanotechnology, analytical characterization plays a vital role in understanding the behavior and toxicity of nanomaterials (NMs). Characterization needs to be thorough and the technique chosen should be well-suited to the property to be determined, the material being analyzed and the medium in which it is present. Furthermore, the instrument operation and methodology need to be well-developed and clearly understood by the user to avoid data collection errors. Any discrepancies in the applied method or procedure can lead to differences and poor reproducibility of obtained data. This paper aims to clarify the method to measure the hydrodynamic diameter of gold nanoparticles by means of Nanoparticle Tracking Analysis (NTA). This study was carried out as an inter-laboratory comparison (ILC) amongst seven different laboratories to validate the standard operating procedure&#8217;s performance and reproducibility. The results obtained from this ILC study reveal the importance and benefits of detailed standard operating procedures (SOPs), best practice updates, user knowledge, and measurement automation.

The protocol described here aims to measure the hydrodynamic diameter of spherical nanoparticles, more specifically gold nanoparticles, in aqueous media by means of Nanoparticle Tracking Analysis (NTA). The latter involves tracking the movement of particles due to Brownian motion and implementing the Stokes-Einstein equation to obtain the hydrodynamic diameter.

In the field of nanotechnology, analytical characterization plays a vital role in understanding the behavior and toxicity of nanomaterials (NMs). ...

Chemistry

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction with lipids employ naked bilayer membranes. Such systems lack the complexities of crowding by membrane-embedded proteins and glycans and exclude the associated volume effects encountered on cellular membrane surfaces. Also, the negatively charged glass surface onto which the lipid bilayers are formed prevents the free diffusion of transmembrane biomolecules. Here, we present a well-characterized polymer-lipid membrane as a mimic for crowded lipid membranes. This protocol utilizes polyethylene glycol (PEG)-conjugated lipids as a generalized approach for incorporating crowders into the supported lipid bilayer&#160;(SLB). First, a cleaning procedure of the microscopic slides and coverslips for performing single-molecule experiments is presented. Next, methods for characterizing the PEG-SLBs and performing single-molecule experiments of the binding, diffusion, and assembly of biomolecules using single-molecule tracking and photobleaching are discussed. Finally, this protocol demonstrates how to monitor the nanopore assembly of bacterial pore-forming toxin Cytolysin A (ClyA) on crowded lipid membranes with single-molecule photobleaching analysis. MATLAB codes with example datasets are also included to perform some of the common analyses such as particle tracking, extracting diffusive behavior, and subunit counting.

Here, a protocol to perform and analyze the binding, mobility, and assembly of single molecules on artificial crowded lipid membranes using single-molecule total internal reflection fluorescence (smTIRF) microscopy is presented.

Cellular membranes are highly crowded environments for biomolecular reactions and signaling. Yet, most in vitro experiments probing protein interaction ...

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, &#34;kiss-and-run&#34; fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore &#34;openness&#34;, the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

Neuroscience

Temperature-Controlled Assembly and Characterization of a Droplet Interface Bilayer

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits versus other methods: DIBs are stable and often long-lasting, bilayer area can be reversibly tuned, leaflet asymmetry is readily controlled via droplet compositions, and tissue-like networks of bilayers can be obtained by adjoining many droplets. Forming DIBs requires spontaneous assembly of lipids into high density lipid monolayers at the surfaces of the droplets. While this occurs readily at room temperature for common synthetic lipids, a sufficient monolayer or stable bilayer fails to form at similar conditions for lipids with melting points above room temperature, including some cellular lipid extracts. This behavior has likely limited the compositions&#8212;and perhaps the biological relevance&#8212;of DIBs in model membrane studies. To address this problem, an experimental protocol is presented to carefully heat the oil reservoir hosting DIB droplets and characterize the effects of temperature on the lipid membrane. Specifically, this protocol shows how to use a thermally conductive aluminum fixture and resistive heating elements controlled by a feedback loop to prescribe elevated temperatures, which improves monolayer assembly and bilayer formation for a wider set of lipid types. Structural characteristics of the membrane, as well as the thermotropic phase transitions of the lipids comprising the bilayer, are quantified by measuring the changes in electrical capacitance of the DIB. Together, this procedure can aid in evaluating biophysical phenomena in model membranes over various temperatures, including determining an effective melting temperature (TM) for multi-component lipid mixtures. This capability will thus allow for closer replication of natural phase transitions in model membranes and encourage the formation and use of model membranes from a wider swath of membrane constituents, including those that better capture the heterogeneity of their cellular counterparts.

This protocol details the use of a feedback temperature-controlled heating system to promote lipid monolayer assembly and droplet interface bilayer formation for lipids with elevated melting temperatures, and capacitance measurements to characterize temperature-driven changes in the membrane.

The droplet interface bilayer (DIB) method for assembling lipid bilayers (i.e., DIBs) between lipid-coated aqueous droplets in oil offers key benefits ...

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

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