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 laye...

<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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#160;mW&#160;</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&#160; gold electrodes are made from 25 mm x 75&#160;mm x&#160;1&#160;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%&#160;</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>&#160;99.9% Pure ethanol</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>34963</td>
			<td>Absolute,&#160; 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 ...

Watch this Scientific Journal Video about Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes at JoVE.com

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 ...

This protocol offers a nanomaterial electrode interface that mimics natural cell membranes making it possible to determine the effect of localized heat transfer from irradiated gold nanoparticles on the integrity of a biological membrane. The nanoparticle and photothermal effects can be explored in real time on model cell membranes. This technique uses a biomimetic membrane system to observe and interrogate photothermal effects on a biological system resulting from the heat generated due to the interaction of gold nanoparticles with light by measuring the conductance and capacitance changes across the model cell membrane.The use of nanoparticles and photothermal therapy is showing great promise as potential cancer therapy. This method provides direct information on how this approach affects cells. This platform provides valuable data to design strategies for thermal therapies that permit optimization of the laser parameters, particle size, particle coatings and composition.Demonstrating the procedure will be UTS researchers, Professor Stella Valanzuela and Dr.Amani Alghalayini along with Professor Bruce Cornell from surgical diagnostics. To begin carefully take off one coplanar gold electrode slide from its container using tweezers taking care to not contact the patterned areas where the tBLMS will form. Air-dry the slide for one to two minutes to remove any residual ethanol.Then place the goal electrode over a dry surface with the patterned gold surface facing up. Peel the transparent adhesive layer cover from a thin laminate and place it over the six channels to define each well. Use a pressure roller to release any air between the slide and the transparent adhesive layer.Within one to two minutes, introduce the second livid bilayer to the assembled first monolayer coated electrode for self-assembly. Add six microliters of three millimolar lipids of interest to the first well of the six well slide without touching the edge of the micropipet tip to the gold surface, which can damage the tethered chemistries on the electrode. Introduce six microliters of the lipid mixture to the other wells with a ten second gap between each edition.Incubate each well for exactly two minutes at room temperature. Exchange the lipid mixture over the electrodes with a buffer such as PBS spacing the times for the addition and buffer exchange 10 seconds apart so that each well is incubated with a lipid for exactly two minutes. Wash the electrodes three more times with 50 microliters of PBS buffer always leaving 50 microliters of buffer over the electrodes and not allowing them to dry.Insert the prepared electrode slide into an AC impedance spectrometer. Make sure that the spectrometer is connected to a computer running the software via USB port. Open the software, click set up and open hardware.Set the hardware settings to use 25 millivolts speak to peak ACX citation and set the frequencies between 0.5 and 10, 000 hertz with two steps per decade for rapid impedance measures. Press okay then click the setup menu and open model. Using an equivalent circuit model that describes the tethering gold electrode as a constant phase element in series with the electrolyte buffer as a resistor and a parallel resistor capacitor network to describe the lipid bilayer, then press okay.Press the start button to start a real-time measurement of membrane capacitance and membrane conduction. After running the protocol and finishing the experiment, save the data. Repeat the measurement with the next well.Use an optics table to set up the experiment to reduce unwanted vibrations and perform experiments in a light proof box to minimize the laser hazard. Lace the impedance reader where the gold side is connected on an XYZ stage. And elevate it such that it sits in the path of the laser source.Use coarse find focusing microscopic gearing to control the height of the laser source to achieve the appropriate position. Target the laser path along the longitudinal axis of the electrode slide and allow the selected tuned laser to stabilize. Before beginning, assess the laser power output using a power meter to ensure only very low wattages are delivered to the tBLMs.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. 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. Lock the knob to secure the position of the laser path when there are no conductance changes observed due to interaction of the laser with the underlying gold electrode.Repair the laser beam light alignment and add gold nanoparticles of interest to the PBS buffer where the tBLMs are immersed while the laser is switched off. The coplanar gold electrode slide with six wells was ultimately defined by the addition of a thin transparent adhesive layer. The tBLM model consists of a spacer and molecules tethered to the gold substrate surface to form the first layer and a non-tether lipids in the second layer.Schematic representation of the different positions of horizontal laser alignment and the normalized conductance recordings over time corresponding to the different alignment positions are shown here. The changes in membrane conductance at positions one and two are due to the heat from interactions of the laser with nanostructures within this buttered gold layer. Focusing the horizontal laser light directly towards the gold electrode causes an increase in membrane conductance.The precise laser position revealed a negligible variation to the membrane conductance recordings during periods of laser on and laser off. The addition of strep divide and conjugated 30 nanometer gold nanoparticles to tBLMs showed a clear difference between the laser on and off periods as well as distinct increases in conduct and Samplitude during the laser on phase. Careful and precise alignment of the laser path towards the buffer surrounding the lipid bilayer membrane is a critical step in this protocol.The thermal predictive model can be used to provide the mathematical determination of the amount of localized heat generated by each GNP. This technique determines the efficiency of heat delivery within biological tissues and highlights the role of targeted irradiated, GNP hyperthermia treatments in vivo.

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

Research

JoVE Journal

Biology

2.5K visualizações.  University of Technology Sydney. This protocol offers a nanomaterial electrode interface that mimics natural cell membranes making it possible to determine the effect of localized heat transfer from irradiated gold nanoparticles on the integrity of a biological membrane. The nanoparticle and photothermal effects can be explored in real time on model cell membranes. This technique uses a biomimetic membrane system to observe and interrogate photothermal effects on a biological system resulting from the heat generated due to t...