Donald J. Leo

Spectral Analysis and Characterization of a Membrane-Based Artificial Hair Cell Sensor

A fully hydrogel-supported, artificial hair cell (AHC) sensor based on bilayer membrane mechanotransduction is designed with sensitivity and versatility in mind. Thanks to fabrication improvements from previous generations, the sensor demonstrates peak current outputs in the nanoamp range and can clearly measure inputs as high as 2k Hz. Characterization of the AHC response to base excitation and air pulses show that membrane current oscillates with the first three bending modes of the hair. Output magnitudes reflect of vibrations near the base of the hair. A 2 DOF Rayleigh-Ritz approximation of the system dynamics yields estimates of 19 N/m and 0.0011 Nm/rad for the equivalent linear and torsional stiffness of the hair’s hydrogel base, although double modes suggest non-symmetry in the gel’s linear stiffness. The sensor output scales linearly with applied voltage (1.79 pA/V), avoiding a higher-order dependence on electrowetting effects. The free vibration amplitude of the sensor also increases in a linear fashion with applied airflow pressure (18.4 pA/psi). Based on these sensitivity characteristics, an array sensing strategy for these sensors is proposed.Copyright

Dynamic Characterization of Biomimetic Artificial Hair Cells

The research presented in this paper investigates the relationship between fluid flow characteristics in an artificial cochlear environment and artificial hair cell sensor response. First, a lipid bilayer-based hair cell sensor is created to model the inner hair cells of the human cochlea. The artificial cochlear environment is then fabricated to recreate the pulsating fluid flow around the artificial inner hair cell stereocilia. Mechanical excitation creates sinusoidal fluid flows in the artificial cochlear environment at a range of frequencies determined by the response of the hair cell sensor in air. For excitation frequencies at and below 40 Hz, the response of the hair cell sensor is approximately equal to the control case having no bilayer. At these low frequencies, bilayer dynamics do not appear to lead to current generation. At frequencies at and above 70 Hz, and in the absence of an externally applied DC offset across the bilayer, the hair cell sensors featuring a bilayer generate up to double the RMS current. Therefore, for excitation frequencies at and above 70 Hz, bilayer dynamics play a significant role in hair cell sensor response. Further testing of the hair cell sensor shows that applying a DC offset across the bilayer increases the peak-to-peak sensor output by up to a factor of 80.Copyright

Encapsulating Networks of Droplet Interface Bilayers in a Thermoreversible Organogel

The development of membrane-based materials that exhibit the range and robustness of autonomic functions found in biological systems remains elusive. Droplet interface bilayers (DIBs) have been proposed as building blocks for such materials, owing to their simplicity, geometry, and capability for replicating cellular phenomena. Similar to how individual cells operate together to perform complex tasks and functions in tissues, networks of functionalized DIBs have been assembled in modular/scalable networks. Here we present the printing of different configurations of picoliter aqueous droplets in a bath of thermoreversible organogel consisting of hexadecane and SEBS triblock copolymers. The droplets are connected by means of lipid bilayers, creating a network of aqueous subcompartments capable of communicating and hosting various types of chemicals and biomolecules. Upon cooling, the encapsulating organogel solidifies to form self-supported liquid-in-gel, tissue-like materials that are robust and durable. To test the biomolecular networks, we functionalized the network with alamethicin peptides and alpha-hemolysin (αHL) channels. Both channels responded to external voltage inputs, indicating the assembly process does not damage the biomolecules. Moreover, we show that the membrane properties may be regulated through the deformation of the surrounding gel.

A Hydrogel-Based Droplet Interface Lipid Bilayer Network

In this work, we present a process for the fabrication of meso-scale hydrogel-based lipid bilayer arrays. The hydrogels support lipid monolayers at an oil-water interface, and when brought together, form stable bilayers. The substrates are formed using 3D printed molds and include built-in, customizable circuits patterned with silver paint. The system can be adapted to varying network sizes and circuit designs, and new arrays are fabricated quickly and inexpensively using common laboratory techniques. An enclosed 3×3 array with 3 mm spacing between neighboring hydrogels and electrodes to individually examine each bilayer has been created using this method. An alternative test setup was also developed to better observe the formation of bilayers in a small array. Using this setup, two bilayers were formed simultaneously, demonstrating the feasibility of this type of system and providing valuable information for expanding and improving the enclosed network. Many of the design concepts presented here can be adapted for use at smaller scales using microfabrication techniques.Copyright

Mechanosensitive droplet interface bilayer networks

A method for studying the coupled electrical-mechanical response of droplet interface bilayers is proposed. This research examines the concept of the biologically-inspired hair cell in greater depth, attempting to determine the source of the sensing current when no external potential is applied across the sensing droplet-interface bilayer element. Historically the mechanosensitive current in these droplet-interface bilayers has been attributed to a combination of capacitive currents and electrode oscillation (experimental error); however the development of a third sensing mechanism through modifying the bilayer properties may enhance the usefulness of the mechanosensitive droplet interface bilayer networks considerably. This would allow for measurable sensing currents without requiring an externally applied electric field by permanently charging the bilayer element through surface modifications. Charging agents are added to the droplet interface bilayer network as the network is oscillated and the electrical response is recorded for analysis. The adsorption of the charged molecules is studied through the intramembrane field compensation (IFC) approach, and the knowledge gained from this is then applied towards the mechanosensitivity analysis. Multiple charging techniques are tested and employed, and the nature of the sensing current is determined by examining the frequency content of the recorded currents. Several properties are derived, including the nature of the sensing current, the charging mechanisms available for boosting the sensing current, and the nature of the sensing current without externally applied potentials.

Directional Sensitivity Analysis of a Hydrogel-Supported Artificial Hair Cell

An artificial hair cell sensor imitates the function of cilia in natural hair cells in order to detect surrounding fluid displacement. Here, a novel structure for creating artificial hair cell sensors uses established methods of creating lipid bilayers at the interfaces of millimeter scale hydrogel shapes. This paper describes the fabrication of the sensor components and the manner in which they are assembled and tested. The hair’s vibration can be detected by monitoring changes in the current produced by mechanical fluctuations in the bilayer. The cross-sectional geometry of the hair can be changed to enable directional sensitivity. Spectral analysis of the sensor current response indicates that frequencies and magnitudes change when a flattened hair is excited in different directions. Finally, the sensor is shown to become more sensitive with applied potential across the bilayer. Results agree with similar studies on this phenomenon.© 2013 ASME

A 3D printing method for droplet-based biomolecular materials

The field of developing biomolecular droplet-based materials using a bottom-up approach remains underexplored. Producing tissue-like materials, from entirely synthetic components, presents an innovative method to reconstruct the functions of life within artificial materials. Aqueous droplets, encased with lipid monolayers, may be linked via bilayer interfaces to make up structures that resemble biological tissues. Here we present the design and development of an easy-to-build 3D printer for the fabrication of tissue-like biomolecular materials from cell-sized aqueous droplets. The droplets are generated using a snap off technique, capable of generating 30 droplets per minute. The printed network of droplets may also be functionalized with various types of membrane proteins to achieve desired engineering applications like sensing and actuation, or to mimic electrical communication in biological systems. Voltage sensitive channels are introduced into selected droplets to create a conductive path with the material in the presence of an external field.

The Effect of Transmembrane Potential on the Gating of MscL Channels in Droplet Interface Bilayers

The ability to functionalize droplet interface bilayers (DIBs) with the MscL channel and its mutants has been demonstrated. In previous work, the V23T gain of function mutant of MscL produced consistent activation when harmonic axial compressions were applied to the aqueous droplets supporting the lipid bilayer, where the channels settle. The deformation of the droplets results, at maximum compression, in an increase in surface area, and thus an increase in tension at the water-lipid-oil interface. This increase in monolayer tension was found to be the product of the relative change in surface area of each of the droplets and the compressibility modulus of the DPhPC monolayer (∼120 mN/m). The tension increase at the water-lipid-oil interface almost doubles to make up the increase in tension in the bilayer interface, resulting in activation of the incorporated MscL channels. However, it was found that the application of a relatively high transmembrane potential (∼100 mV), from an external power source, is a requirement for the activation of the V23T-MscL channels. Here, we investigate and analyze the impact of transmembrane potential on the activity of MscL channels in both a droplet interface bilayer system and E. coli spheroplast via patch-clamp. We demonstrate that the channels became more susceptible to gating upon the application of a negative potential, compared to when a positive potential is applied, proving their sensitivity to voltage polarity.Copyright

Airflow Sensing With Arrays of Hydrogel Supported Artificial Hair Cells

The hair cell is a biological sensor that uses microscopic hair-like structures to detect delicate motions of surrounding fluid. Inspired by this principle, we have created an artificial hair cell (AHC) sensory method based on biomolecular transduction for sensing spatial variations in air flow. The key feature of this method is the use of one-dimensional arrays built from modular AHC units which measure local velocity at different points in a flow profile. Each of the AHC units uses thinly extruded glass fibers as mechanical receptors of air velocity. Hair vibrations are converted to current via hydrogel-supported lipid bilayer membranes through their mechanocapacitive properties. Preliminary tests with linear arrays of three AHC units attempt to measure the air source profile with varying position and intensity. Each unit was fabricated with a hair of different length, giving it a unique vibrational response. This technique was inspired by how organisms use hair cells with tuned responses to mechanically process flow stimuli. A significant challenge in processing the sensors’ output was the limitation of one input channel on the current measurement unit, thus each sensor output had to be sent over the same channel. When several AHC units are excited simultaneously by an airflow, the resulting signal is a superposition of each sensor’s individual response. To separate the signals back into their individual measurements, the Hair Frequency Response Decomposition method is developed, which maps the spectral content of a combined output to the location of excitation in the array. This method takes advantage of the AHC’s high signal-to-noise ratio (compared to other membrane-based AHCs) and linear output response to flow velocity. Results show that the bilayers’ consistent spectral responses allow for an accurate localization of sensor excitation within the array. However, temporal variations in bilayer size affect sensitivity properties and make accurate flow velocity estimation difficult. Nevertheless, under stable bilayer conditions the measured velocity profiles matched closely with theoretical predictions. The implementation of the array sensing method demonstrates the sensory capability of bilayer-based AHC arrays, but highlights the difficulties of achieving consistent performance with bio-molecular materials.Copyright