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 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 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 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 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 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/µm2.
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.
1. tBLMs electrodes preparation
2. Laser irradiation
3. Statistical data analysis and presentation
4. Predict the amount of localized heat generated in the tBLMs from irradiated nanoparticles (thermal predictive model)
The gold substrate upon which tBLMs can be created is shown in Figure 1. A schematic of the experimental setup is presented in Figure 2.
Coplanar gold electrodes, as shown in 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 direct exposure of the laser light to tBLMs membrane. Each well of the electrode array contains a circle-shaped working electrode (area: 0.707 cm2) and half-circle shaped counter electrode or coplanar electrode (area: ~ 0.725 cm2), which are separated by a gap of ~2 mm. The transparent adhesive layer insulates the rest of the deposited gold from the bulk electrolyte. In contrast, the underlying gold layout connects the working electrodes to contact areas outside the measuring chambers to provide the electrical connection to the EIS reader without the need for a reference electrode.
The laser path is aligned in a manner where it is interacting with the tBLMs and is scattered through the liquid buffer surrounding it, but not such that it can interact with the underlying gold substrate. This is easily determined via horizontal raising and lowering of the laser until the correct position is established. This position is just at the point where no changes in membrane conductance can be observed. Given that tBLMs are formed by attachment to a substrate layer of bulk gold, it seems likely that the changes in membrane conductance at position 1 and 2 in Figure 2 are as a result of heat from interactions of the laser with nanostructures within the sputtered bulk gold layer. Thus, using the accurate position of horizontal light beam alignment focusing on eliminating interaction between the laser light and the bulk gold substrate found below the tBLMs.
Focusing the horizontal laser light directly towards the gold electrode causes an increase in membrane conductance, as presented in Figure 2, position 1 and 2. The precise laser position revealed negligible variation to the membrane conductance recordings during both periods of laser ON and laser OFF (Figure 2B, position 3). The GNP sample was added after establishing baseline recordings, as shown in Figure 2, position 3. The addition of streptavidin-conjugated 30 nm gold nanoparticles to tBLMs that contained biotinylated cholesterol showed a clear difference between the laser ON and OFF periods, as well as in comparison to position 3, with distinct increases in conductance amplitude during the laser ON phase (Figure 2B, position 4).
Figure 1: Schematic representation of the tethered bilayer lipid membrane (tBLM) model on a gold substrate. (A) Coplanar gold electrode slide with six wells, ultimately defined by the addition of a thin transparent adhesive layer. (B) The tBLM model comprises spacer (ethylene glycol chains ended with a hydroxyl group) and tethered molecules (ethylene glycol groups ended with hydrophobic phytanyl chain) tethers to the gold substrate surface to form the first layer. The second layer includes the non-tethered lipids. The modified figure was based on Cornell et al.24. Please click here to view a larger version of this figure.
Figure 2: Illustration of the assay set-up for alignment and corresponding measuring membrane conductance changes across tBLMs arising from laser illumination ( λ = 530 nm). (A) Schematic representative of the different positions of horizontal laser alignment; where Position 1: laser light beam aligned with the gold substrate (when the laser was turned ON is indicated in red); position 2 the horizontal laser light mixed with membrane and gold substrate; position 3 laser light focused into the bulk fluid surrounding tBLMs; Position 4 laser beam light focused into the fluid surrounding the tBLMs in the presence of streptavidin-conjugated 30 nm spherical GNPs. (B) Normalized conductance recordings over time correspond to the different alignment positions. Positions 1, 2 and 3 measurements of tBLMs conductance in the absence of GNPs, whereas position 4 is a measurement of tBLMs conductance in the presence of streptavidin-conjugated 30 nm spherical GNPs. The membrane conduction values were normalized to the initial value of membrane conduction upon tBLMs formation. Results are representative of at least three independent experiments. Please click here to view a larger version of this figure.
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 interaction. There is a critical step in this protocol, which is the careful and precise alignment of the laser path towards the buffer surrounding bilayer lipid membrane.
The use of the tBLM model offers distinct electrical sealing properties that mimic natural lipid membranes characteristics24. tBLMs also provide an aqueous ionic reservoir region between the gold substrate and the subsequently formed membrane, where the tethered molecules and the spacer molecule had a thickness of 11 Å25, and the bilayer lipid membrane thickness was around 6.5 nm19. This can offer space to incorporate membrane proteins, ion channels or other specific functionalized molecules13,22. The selection of 70% DPEPC and 30% GDPE lipids provides optimal sealing of bilayer lipid membrane to examine the electrical characteristics of tBLMs using EIS system24. Likewise, the introduction of cholesterol within the bilayer lipid membranes closely mimics native biomimetic model membranes. Cholesterol moieties improve the bilayer lipid membrane stability, as well as minimizing the membrane permeability to ions by providing high packing of the phospholipid bilayer26,27. Combining tBLMs with the EIS system provides indirect measurement of heat transfer between irradiated GNPs and bilayer lipid membranes. Further, the use of coplanar gold electrodes in this protocol enables the real-time EIS measurements without any interference from reference or counter electrodes.
Gold in the nanoparticle scale has different physical and optical characteristics to larger gold aggregates. The size and shape of nanoparticle access their bio-distribution, circulation lifetime and cell uptake, where nanoparticles of intermediate sizes (20-60 nm) exhibit maximum cell uptake as well as offer a high surface area to volume ratio, allowing for subsequent functionalization28,29. The implemented 30 nm GNP size in this study represented intermediate GNPs sizes, while the laser wavelength selection was according to the absorption peak of GNPs to yield the most efficient excitation, which consequently leads to heating. The laser illumination of tBLMs gold surfaces elevates membrane conduction peaks at the laser ON phase. This is proposed to be as a result of bulk gold surface nanostructures that interact with the laser, which would mask heat production phenomena following the addition of the GNPs30. To overcome this, the developed approach here GNPs are illuminated by using horizontal laser alignment across the lipid-buffer interface, as illustrated in Figure 2, position 3 and 4.
The protocols described here can be modified readily by altering the lipid composition of the membrane to mimic various natural cell types, or by altering the introduced GNPs size and shape such as 100 nm gold nanourchins with the corresponding laser beam light19. This can then be used to determine the impact of localized GNPs induced radiation on specific cell types.
In summary, this protocol serves as a robust detection biosensor to study interactions of in situ irradiated GNPs with model bilayer lipid membrane entities of interest to answer questions on heat transfer phenomena. This will assist in developing more efficient photothermal therapies, as well as providing valuable information for detailed mechanisms of heat transfer within biological systems. This approach can be used as a tool for the prediction of the level of cell membrane destruction that can be experienced by these heated nanoparticles.
The authors have nothing to disclose.
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).
30 nm diameter streptavidin-conjugated gold nanoparticles | Cytodiagnostics | AC-30-04-05 | This is a streptavidin-conjugated GNPs product ready for use |
30 nm diameter bare gold nanoparticles | Sigma-Aldrich | 753629 | This is a bare GNPs product ready for use |
Cholesterol-PEG-Biotin (MW1000) | NANOCS | PG2-BNCS-10k | Dissolved in highly pure ethanol |
C20 Diphytanyl-Glycero-Phosphatidylcholine lipids | SDx Tethered Membranes Pty. Ltd. | SDx-S1 | 1 ml glass vial containing 70% C16 diphytanyl phosphatidylcholine (DPEPC) and 30% C16 diphytanyl glycerol (GDPE) in 99.9% ethanol |
Benzyl-disulfide-tetra-ethyleneglycol-OH | SDx Tethered Membranes Pty. Ltd. | SDx-S2 | Spacer molecules |
Benzyl-disulfide (tetra-ethyleneglycol) n=2 C20-phytanyl | SDx Tethered Membranes Pty. Ltd. | SDx-S2 | Tethered molecules |
532 nm green laser continuous light | OBIS LS/OBIS CORE LS, China | ND-1000 | The power of this laser was ~135 mW |
tethaPod EIS reader | SDx Tethered Membranes Pty. Ltd. | SDx-R1 | A reader of conductance and capacitance on six channels simultaneously |
tethaPlate cartridge assembly | SDx Tethered Membranes Pty. Ltd. | SDx-BG | Materials to attach the slide with electrodes to the flow cell cartridge |
Clamp and slide assembly jig | SDx Tethered Membranes Pty. Ltd. | SDx-A1 | Materials to attach the slide with electrodes to the flow cell cartridge |
Lipid coated coplanar gold electrodes | SDx Tethered Membranes Pty. Ltd. | SDx-T10 | Coplanar gold electrodes are made from 25 mm x 75 mm x 1 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% |
tethaQuick software | SDx Tethered Membranes Pty. Ltd. | SDx-B1 | Software for use with tethaPod to process data and display conductance, impedance and capacitance measurements from the tethaPlate electrodes |
99.9% Pure ethanol | Sigma-Aldrich | 34963 | Absolute, 99.9% |
Phosphate buffered saline (PBS) | Sigma-Aldrich | P4417 | pH 7 |