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, 122 (5), 2016-2026

NanoTouch: Intracellular Recording Using Transmembrane Conductive Nanoparticles

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NanoTouch: Intracellular Recording Using Transmembrane Conductive Nanoparticles

Mitsuyoshi L Saito. J Neurophysiol.

Abstract

Observations of the electrophysiological properties of cells are important for understanding cellular functions and their underlying mechanisms. Short action potentials in axons are essential to rapidly deliver signals from the neuronal cell body to the terminals, whereas longer action potentials are required for sufficient calcium influx for transmitter release at the synaptic terminals and for cardiomyocyte and smooth muscle contractions. To accurately observe the shape and timing of depolarizations, it is essential to measure changes in the intracellular membrane potential. The ability to record action potentials and intracellular membrane potentials from mammalian cells and neurons was made possible by Ling and Gerard's discovery in 1949, when they introduced sharp glass electrode with a submicron sized tip. Because of the small tip size, the sharp glass electrode could penetrate the cell membrane with little damage, which was one of the major breakthroughs in cellular electrophysiology and is the basic principle of the intracellular recording technique to date, providing the basis for further innovation of patch-clamp electrophysiology. I report a proof-of-principle demonstration of a novel method for recording intracellular potentials without penetrating the cell membrane using glass electrodes. We discovered that magnetically held transmembrane conductive nanoparticles can function as an intracellular electrode to detect transmembrane membrane potentials similar to those obtained by the conventional patch-clamp recording method.NEW & NOTEWORTHY To accurately observe the shape of action potentials, it is essential to perform intracellular recordings. I present a method to record intracellular potentials using magnetically held magnetic conductive nanoparticles in the membrane as an electrode. These nanoparticles function similarly to a conventional intracellular microelectrode. This is the first report to apply conductive nanoparticles to detect action potentials in the form of electrical signals.

Keywords: conductive nanoparticles; intracellular recordings; needleless electrophysiology.

Conflict of interest statement

This work was submitted under Patent application WO/2018/199334, “Intracellular recording method using the membrane penetrating nanoparticles as an electrode (nanoelectrode).”

Figures

Fig. 1.
Fig. 1.
Conformation of a conventional intracellular recording method and the concept of NanoTouch: gold-coated magnetic nanoparticles (GMNPs) are integral to the intracellular recording method. A: conformation of conventional intracellular recording. A glass microelectrode is inserted into the cell. Outputs from the electrode and the reference electrode placed in the bath are fed to the amplifier (PreAmp), which allows detection of membrane potential changes. B: the concept of “NanoTouch”: a GMNP-based intracellular recording method. An intracellular recording configuration using a nanoparticle intracellular voltage sensor was assembled and comprised 1) polyethylene glycol-stabilized GMNPs (50 nm in diameter), 2) a neodymium magnet (Moritoku Corp.), and 3) a conductive glass slide (CG; 2.5 cm × 2.5 cm, fluorine-doped tin oxide; Kenis) that functions as a cell plating substrate and as a connection material to connect GMNPs with the amplifier when GMNPs penetrate the bottom cell membrane and make contact with (touch) the CG. C: cells were plated on a CG slide and GMNPs were delivered to the cytosol using polyethyleneimine (PEI; dendrimers). Activated dendrimer (SuperFect, Qiagen) or pore-forming peptides (streptolysin O, SLO) can be used in place of PEI (Lévy et al. 2010). Prior to this step, it is important to confirm that the plated cells have formed a sheet, because any gaps in the cell sheet will cause shunt circuits between the bath medium and the CG surface. Next, a magnet was placed below the CG recording chamber, which pulled GMNPs toward the bottom of the cells, which had adhered to the CG. Finally, as GMNPs penetrated and spanned the membrane, the electrical connection between the intracellular compartment of the cell and the CG substrate was monitored with a conventional patch-clamp amplifier. With the reference electrode set in the bath solution and the CG connected to the amplifier inputs, the GMNPs functioned as an intracellular electrode by bridging the cellular membrane and connecting the cytosolic compartment to the CG surface (B).
Fig. 2.
Fig. 2.
Detection of the resting membrane potential and of light-induced channelrhodopsin (ChRWR) depolarization using the NanoTouch recording method. A: detection of the resting membrane potential. Recordings before and after the magnet was placed under the conductive glass slide (CG) are shown. Placing the magnet under the CG triggered very slow downward movement. Without the magnet, this did not occur, indicating that for the gold-coated magnetic nanoparticles (GMNPs) to become transmembrane, a magnetic pulling force is required and it does not occur spontaneously. B: ChRWR responsiveness to blue light stimuli was validated using the patch-clamp technique. Under current-clamp mode, membrane potential was controlled at −40 mV. Light strength was increased from 0.2 to 0.8 A in 0.2-A intervals (25, 50, 75, 100% power), and the membrane voltage responses were recorded, which increased as stimulus strength increased (left). Normalized results (V/Vmax × 100%) are shown in box and whisker plot (right), with the maximum response as 100% (n = 4). The maximum LED power used gave 7.4 ± 1.8 mV at −40-mV depolarization. C: NanoTouch recording of blue light activation of ChRWR. ChRWR-expressing CHO cells were subjected to blue light stimulation as in the patch-clamp method. Light strength was increased as in B, and the membrane voltage responses were recorded (left). Light strength-dependent ChRWR responses are shown in box and whisker plot (right), with the maximum response as 100%. The maximum LED power used gave depolarization of 5.1 ± 3.1 mV (n = 4; resting membrane potential was calculated to be −33 mV). This demonstrates that the GMNPs functioned similarly to a conventional intracellular microelectrode. D: another example of NanoTouch recording of blue light activation of ChRWR. Light strength was increased as in B and was followed by 5 repetitions of brief light pulses (750 ms on, 750 ms off).
Fig. 3.
Fig. 3.
Validation of Nav1.5/Kir2.1 human embryonic kidney (HEK) cells under voltage-clamp and recording comparisons of patch-clamp and NanoTouch methods. A: depolarization voltage pulses resulted in transient inward current typical of the Nav1.5 current (top left), and the current-voltage (I-V) relationship [4,250.6 ± 499 pA (n = 4) at −20 mV] was plotted (top right). Hyperpolarizing voltage pulses resulted in inward rectifying current typical of the Kir2.1 current. It was completely blocked by barium (50 μM), an inward rectifier blocker (bottom left). The I-V plot was generated and had an amplitude of 760 ± 13.8 pA (n = 4) at −120 mV (bottom right). B: passive and active membrane responses using the patch-clamp. A series of depolarizing current pulses was applied and resulted in electrotonic depolarizing responses (left). As the injection current (I) amplitude was raised, a regenerative component appeared (left, 3rd trace). The I-V relationship was plotted (right), and passive membrane resistance (PMR) was 719.6 ± 1.7 MΩ (n = 4). Cell capacitance was also calculated by fitting the raw voltage traces. Single-cell capacitance values were 7.0 ± 1.8 pF (n = 5). C: passive and active membrane responses using NanoTouch. Passive membrane properties of the HEK cell sheet were examined using the NanoTouch method under current clamp. In response to a series of depolarizing current injections, passive membrane depolarizations were observed. Their voltage deflections increased as the applied current increased. In some cases, the voltage response took the regenerative form (left). The I-V relationship (n = 4) (right) gave a PMR of 1.34 ± 0.19 GΩ and total capacitance of 2.27 ± 0.23 μF (n = 3). Vm, resting membrane potential (arrows).
Fig. 4.
Fig. 4.
Recording Nav1.5/Kir2.1 human embryonic kidney (HEK) cell action potentials using manual patch-clamp and NanoTouch methods. A: all-or-nothing-type action potentials, anodal break action potentials, were generated in Nav1.5/Kir2.1 cells under current clamp. As prepulse-induced hyperpolarization became larger, the subsequent anodal break action potential amplitudes also increased, indicating that Nav1.5 inactivation was removed by the hyperpolarizing prepulse. Voltage (mV) indicates the prepulse membrane potentials. The arrows indicate the expanded views of action potentials in the middle trace. B: action potentials recorded using patch-clamp (broken line) and NanoTouch (solid line) methods were superimposed by adjusting their amplitude and indicate almost identical forms. C: NanoTouch captures spontaneous action potentials. The spontaneous generation of action potentials lasted 30 min. Action potential amplitudes were 39 ± 1.5 mV (n = 85 action potentials analyzed), and the arrow indicates Vm (top trace). Bottom trace shows an expanded view of the action potentials. D: NanoTouch recordings shown in C were further subjected to current stimulation. In response to the current injection (I), the membrane depolarized much quicker than the recordings shown in Fig. 3C, but they failed to trigger action potentials (spontaneous action potentials appeared irrespective of current injection).
Fig. 5.
Fig. 5.
Schematic illustration of the conductive nanoparticle-magnet electrode (MagEle) recording method and application of MagEle recording to differentiated NG-108-15 hybridoma cells. A: the MagEle mode of the NanoTouch method utilizes a conductive nickel-coated neodymium magnet as an electrode (a conductive material equivalent to the conductive glass used in NanoTouch). Gold-coated magnetic nanoparticles (GMNPs) were first introduced to target cells using polyetherimide, and then the magnet was pressed directly against cells to pull GMNPs upward. Once the nickel-coated neodymium magnet made contact with the GMNPs, membrane potential changes were detected. Therefore, the magnet itself functions as a connecting material to the amplifier input. B: characterization of differentiated NG108-15 cells using the patch-clamp method under current clamp. Differentiated NG108-15 cells responded to depolarizing current injection and generated action potentials (B1). NG108-15 cells generate almost no spontaneous activity; therefore, 20 μM veratridine (a sodium channel opener) was used to activate NG108-15 cells. On application of veratridine, spontaneous action potentials appeared (B2). C: MagEle recording of veratridine-activated differentiated NG108-15 cell action potentials. Veratridine (20 μM) was used to activate NG108-15 cells to induce spontaneous action potentials. The region indicated was expanded to show the shapes of NG108-15 cell action potentials. Veratridine not only induced spontaneous action potentials but also triggered bursting activities that were not seen in electrically induced action potentials in the patch-clamp study, and they lasted for 90 min. Relative voltage units (R.V.U.) were used to show action potential amplitude.

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