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ref: -0 tags: nanoprobe transmembrane intracellular thiol gold AFM juxtacellular date: 02-06-2017 23:45 gmt revision:3 [2] [1] [0] [head]

PMID-20212151 Fusion of biomimetic stealth probes into lipid bilayer cores

  • Used e-beam evaporation of Cr/Au/Cr 10/10/10 or 10/5/10 onto a Si AFM tip.
    • Approx 200nm diameter; 1800 lipid interaction at the circumference.
  • Exposed the Au in the sandwich via FIB
  • Functionalized the Au with butanethiol or dodecanthiol; former is mobile on the surface, latter is polycrystaline.
    • Butanethiol showed higher adhesion to the synthetic membranes
  • Measured the penetration force & displacement through synthetic multi-layer lipid bilayers.
    • These were made via a custom protocol with 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) and cholesterol

PMID-21469728 '''Molecular Structure Influences the Stability of Membrane Penetrating Biointerfaces.

  • Surprisingly, hydrophobicity is found to be a secondary factor with monolayer crystallinity the major determinate of interface strength
  • Previous studies using ellipsometry and IR spectroscopy have shown that alkanethiol self-assembled monolayers display an abrupt transition from a fluid to a crystalline phase between hexanethiol and octanethiol.
    • This suggests the weakening of the membrane stealth probe interface is due to the crystallinity of the molecular surface with fluid, disordered monolayers promoting a high strength interface regime and rigid, crystalline SAMs forming weak interfaces.

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ref: -0 tags: intracellular juxtacellular recording tungsten nanowire whole cell patch date: 02-06-2017 22:39 gmt revision:2 [1] [0] [head]

PMID-22905231 Neuronal recordings with solid-conductor intracellular nanoelectrodes (SCINEs).

  • <300 nm diameter W fibers, several um long, fabricated via FIB.
  • Functionalized with a hydrophobic silane on the oxide.
    • Quite complete & custom methods here.
  • Not quite whole cell recording, but excellent SNR; 4mv APs.
    • Slice, rat hippocampus organotypic.
    • Expected much larger recorded APs; suspect partial membrane penetration.
    • Only lasted a few seconds to minutes.
  • Needed custom recording setup for interfacing with 100Gohm electrodes; stray capacitance < 4 pf.
  • Intracellular electrodes must be designed to not shunt the membrane open upon insertion.
    • In a study where whole-cell recordings were established prior sharp microelectrode penetration, all neurons showed significant depolarization following impalement.
    • Here there was no change in membrane voltage in 10% of insertions of the silane-functionalized SCINEs. only in the functionalized electrodes).
    • Minor distortion of the AP was observed.
  • In whole-cell patch clamping, diffusion from the pipette to the cytosol interrupts biochemical processes necessary for normal cellular function (e.g. respiration!).
  • The hardness of the tungsten ensures that SCINEs can be repeatedly inserted millimeter-deep into brain tissue without noticeable damage to the tip.
    • E.g. 300 nm tungsten will not easily navigate vasculature...

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ref: -0 tags: juxtacellular recording gold mushroom cultured hippocampal neurons Spira date: 02-01-2017 02:44 gmt revision:7 [6] [5] [4] [3] [2] [1] [head]

Large-Scale Juxtacellular Recordings from Cultured Hippocampal Neurons by an Array of Gold-Mushroom Shaped Microelectrodes

  • Micrometer sized Au mushroom MEA electrodes.
  • Functionalized by poly-ethylene-imine (PEI, positively charged)/laminin (extracellular matrix protein) undergo a process to form juxtacellular junctions between the neurons and the gMµEs.
  • No figures, but:
    • Whereas substrate integrated planar MEA record FPs dominated by negative-peak or biphasic-signals with amplitudes typically ranging between 40-100 µV and a signal to noise ratio of ≤ 5,
    • The gMµE-MEA recordings were dominated by positive monophasic action potentials.
    • It is important to note that monophasic high peak amplitudes ≥ 100 µV are rarely obtained using planar electrodes arrays, whereas when using the gMµE-MEA, 34.48 % of the gMµEs recorded potentials ≥ 200 µV and 10.64 % recorded potentials in the range of 300-5,085 µV.
  • So, there is a distribution of coupling, approximately 10% "good".

PMID-27256971 Multisite electrophysiological recordings by self-assembled loose-patch-like junctions between cultured hippocampal neurons and mushroom-shaped microelectrodes.

  • Note 300uV - 1mV extracellular 'juxtacellular' action potentials from these mushroom recordings. This is 2 - 5x better than microwire extacellular in-vivo ephys; coupling is imperfect.
    • Sharp glass-insulated W electrodes, ~ 10Mohm, might achieve better SNR if driven carefully.
  • 2um mushroom cap Au electrodes, 1um diameter 1um long shaft
    • No coating, other than the rough one left by electroplating process.
    • Impedance 10 - 25 Mohm.
  • APs decline within a burst of up to 35% -- electrostatic reasons?
  • Most electrodes record more than one neuron, similar to in-vivo ephys, with less LFP coupling.

PMID-23380931 Multi-electrode array technologies for neuroscience and cardiology

  • The key to the multi-electrode-array ‘in-cell recording’ approach developed by us is the outcome of three converging cell biological principals:
    • (a) the activation of endocytotic-like mechanisms in which cultured Aplysia neurons are induced to actively engulf gold mushroom-shaped microelectrodes (gMμE) that protrude from a flat substrate,
    • (b) the generation of high Rseal between the cell’s membrane and the engulfed gMμE, and
    • (c) the increased junctional membrane conductance.
  • Functionalized the Au mushrooms with an RGD-based peptide
    • RGD is an extracellular matrix binding site on fibronectin, which mediates it's interaction with integrin, a cell surface receptor; it is thought that other elements of fibronectin regulate specificity with its receptor. PMID-2418980

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ref: -0 tags: vertical nanowire juxtacellular recording date: 02-01-2017 00:50 gmt revision:2 [1] [0] [head]

PMID-22231664 Vertical nanowire electrode arrays as a scalable platform for intracellular interfacing to neuronal circuits.

  • Note actual coupling is low, 0.002, compared to patch-clamp (400uV vs 200mV). Signal is rather noisy.
  • Dissociated cultures of rat cortical neurons
  • Stimulation current 200 pa enough to change membrane potential, but not initiate a spike.
    • This is 200e-12 / 20e-6 = 5 orders of magnitude lower current than typical ICMS.