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.
