Flow characterization and patch clamp dose responses using jet microfluidics in a tubeless microfluidic device.

BACKGROUND: Surface tension passive pumping is a way to actuate flow without the need for pumps, tubing or valves by using the pressure inside small drop to move liquid via a microfluidic channel. These types of tubeless devices have typically been used in cell biology. Herein we present the use of tubeless devices as a fluid exchange platform for patch clamp electrophysiology. NEW METHOD: Inertia from high-speed droplets and jets is used to create flow and perform on-the-fly mixing of solutions. These are then flowed over GABA transfected HEK cells under patch in order to perform a dose response analysis. RESULTS: TIRF imaging and electrical recordings are used to study the fluid exchange properties of the microfluidic device, resulting in 0-90% fluid exchange times of hundreds of milliseconds. COMSOL is used to model flow and fluid exchange within the device. Patch-clamping experiments show the ability to use high-speed passive pumping and its derivatives for studying peak dose responses, but not for studying ion channel kinetics. COMPARISON WITH EXISTING METHOD(S): Our system results in fluid exchange times slower than when using a standard 12-barrel application system and is not as stable as traditional methods, but it offers a new platform with added functionality. CONCLUSIONS: Surface tension passive pumping and tubeless devices can be used in a limited fashion for electrophysiology. Users may obtain peak dose responses but the system, in its current form, is not capable of fluid exchange fast enough to study the kinetics of most ion channels.

Subangstrom single-molecule measurements of motor proteins using a nanopore.

Techniques for measuring the motion of single motor proteins, such as FRET and optical tweezers, are limited to a resolution of ∼300 pm. We use ion current modulation through the protein nanopore MspA to observe translocation of helicase Hel308 on DNA with up to ∼40 pm sensitivity. This approach should be applicable to any protein that translocates on DNA or RNA, including helicases, polymerases, recombinases and DNA repair enzymes.

Creation and regulation of ion channels across reconstituted phospholipid bilayers generated by streptavidin-linked magnetite nanoparticles.

In this work, we explore the nature of ion-channel-like conductance fluctuations across a reconstituted phospholipid bilayer due to insertion of ∼100 nm sized, streptavidin-linked magnetite nanoparticles under static magnetic fields (SMFs). For a fixed bias voltage, the frequency of current bursts increases with the application of SMFs. Apart from a closed conductance state G(0) (≤14 pS), we identify four major conductance states, with the lowest conductance level (G(1)) being ∼126 pS. The number of channel events at G(1) is found to be nearly doubled (as compared to G(0)) at a magnetic field of 70 G. The higher-order open states (e.g., 3G(1), 5G(1)) are generally observable at larger values of biasing voltage and magnetic field. When the SMF of 145 G is applied, the multiconductance states are resolved distinctly and are assigned to the simultaneous opening and closing of several independent states. The origin of the current bursts is due to the instantaneous mechanical actuation of streptavidin-linked MNP chains across the phospholipid bilayer. The voltage-controlled, magnetogated ion channels are promising for diagnoses and therapeutic applications of excitable membranes and other biological systems.

Rapid fabrication and piezoelectric tuning of micro- and nanopores in single crystal quartz.

We outline the fabrication of piezoelectric through-pores in crystalline quartz using a rapid micromachining process, and demonstrate piezoelectric deformation of the pore. The single-step fabrication technique combines ultraviolet (UV) laser irradiation with a thin layer of absorbing liquid in contact with the UV-transparent quartz chip. The effects of different liquid media are shown. We demonstrate that small exit pores, with diameters nearing the 193 nm laser wavelength and with a smooth periphery, can be achieved in 350 µm thick quartz wafers. Special crater features centring on the exit pores are also fabricated, and the depth of these craters are tuned. Moreover, by applying a voltage bias across the thickness of this piezoelectric wafer, we controllably contract and expand the pore diameter. We also provide a sample application of this device by piezoelectrically actuating alamethicin ion channels suspended over the deformable pore.

On-chip stochastic resonance of ion channel systems with variable internal noise.

We induced stochastic resonance in planar lipid bilayer systems with alamethicin ion channels, and varied alamethicin concentration, membrane area, and applied voltage. We found that membrane-induced microphonic noise significantly affects the signature of stochastic resonance, and that this noise can be used to optimize ion channel-based biosensors.

Mechanical actuation of ion channels using a piezoelectric planar patch clamp system.

High-throughput screening of ion channels is now possible with the advent of the planar patch clamp system. This system drastically increases the number of ion channels that can be studied, as multiple ion channel experiments can now be conducted in parallel. However, due to tedious, usually pressure-driven mechanotransduction techniques, there has been a slow integration of this technology into the field of mechanosensitive ion channels. By implementing a piezoelectric quartz substrate into a planar patch clamp system, we show that the patch clamp substrate itself can be used to mechanically actuate ion channels. The piezoelectric substrate transduces an external, applied electric field into a mechanical tension, so precise actuation of the membrane can be accomplished. By applying this electric field only to the outer edges of the substrate, no ulterior electric field is created in the vicinity of the membrane during actuation. Further, with resonant frequencies ranging from 1 kHz to 200 MHz, quartz substrates can be used to apply a wide range of time-varying tensions to cell membranes. This will allow for new and instructive investigations into the dynamic mechanotransductive properties of ion channels.

Single-ion channel recordings on quartz substrates.

We show that a single-crystal quartz substrate provides a working platform for ion channel research. Single-crystal quartz is piezoelectric, so it can be nanomechanically actuated to perform precise membrane deformations. This, along with its superior noise properties, makes single-crystal quartz ideal for analyzing mechanosensitive ion channels.