Team:Pasteur Paris/Membrane

""

MEMBRANE

When manipulating genetically engineered organisms, it is crucial to guarantee the confinement of these organisms. In our case, we want the genetically modified bacteria to stay at the interface between the prosthesis and the external organic medium. At the same time, one of the main issues our project wants to tackle is the conduction of the neuron influx to the prosthesis. The answer to these questions came as a double solution: confinement of the bacteria by conductive nanoporous membranes. The membrane’s nanoporosity allows substances produced by our modified biofilm to pass through the membrane, but the bacteria remain confined. We tested the conductivity and biocompatibility of two types of membranes.

Figure 1: Bacteria + Conductive Nanoporous Membrane = Confined Bacteria

Gold-coated membranes

Sterlitech Polycarbonate Gold-Coated Membrane Filters were the first membranes we tested. The pores have a diameter of 0.4 micrometer, which is small enough to confine Escherichia coli bacteria, which diameter and size are respectively about 1 micrometer and 2 micrometers. These membranes were relatively easy to manipulate with a forceps because of their high flexibility.

Figure 2: Gold-Coated Membrane
Figure 3: Gold-Coated Membrane (Electron Microscope)

Polymer-coated membranes

The other membranes were Sterlitech Alumina Oxide Membrane Filters with 0.2 micrometer pores. Their higher rigidity compared to the gold-coated membranes led to several membranes being broken while manipulating them with a forceps. We used these membranes as a support for different conductive and biocompatible polymers: PEDOT:PSS (poly(3,4-ethylenedioxythiophene) polystyrene sulfonate), PEDOT:Cl and PEDOT:Ts.

Figure 4: Alumnia Oxyde Membrane in grey
Figure 5: Alumnia Oxyde Membrane in grey (electron microscope)

For PEDOT:PSS, an aqueous solution of PEDOT:PSS was prepared ([1]) and alumina oxide membranes were dipped for 24 hours in this solution. Electron microscopy of the membranes before and after the experiment showed the deposit of a substance on their surface; however its nature has not been tested.

Figure 6: PEDOT:PSS-coated membrane
Figure 7: PEDOT:PSS-coated membrane (electron microscope)

Vapor-phase polymerization of PEDOT:Cl and PEDOT:Ts ([2]) also induced a change in the surface of the membranes (its exact nature also has not been verified).

Figure 8: PEDOT:TS (left) / PEDOT:CL (right) - coated membranes
Figure 9: PEDOT:CL - coated membrane (electron microscope)

Biocompatibility

The first issue to tackle for such an interface is its biocompatibility, so its ability to coexist with a living organism. Experiments in self-made PDMS culture wells with E. coli showed a low biocompatibility for the gold coated membrane, but an enhanced biocompatibility for the polymer-coated membranes.

Conductivity

The second criterion for a fully functional interface is its ability to conduct a neuron’s influx. Thus, conductivity measurements were made for signals of different frequencies on the membranes. Results showed excellent conductive properties for the gold-coated membranes and very good conductive properties for the polymer-coated membranes.

CONCLUSION

Biocompatible polymers like PEDOT:PSS represent ideal materials for engineering biocompatible and conductive interfaces, that are also relatively easy to produce, thus making them our preferred choice in our project. However, it is worth mentioning that we are totally aware of the fact that we can’t just expect neuron axons to bind to our interface and produce an electric signal. The electric signal transmitted by a nerve is heavily limited to the interior of the nerve by myelin covering the axon, and the signal transmitted by the axon is purely chemical. So it requires special electrodes, like Fine or Cuff electrodes, to detect an electric signal. We might explore these solutions in the continuation of our project to enhance our interface’s ability to transmit neuron signals.

REFERENCES

  • Jikui Wang, Guofeng Cai, Xudong Zhu, Xiaping Zhou, Oxidative Chemical Polymerization of 3,4-Ethylenedioxythiophene and its Applications in Antistatic coatings, Journal of Applied Polymer Science, 2012, Vol. 124, 109-115 .

  • Alexis E. Abelow, Kristin M. Persson, Edwin W.H. Jager, Magnus Berggren, Ilya Zharov, Electroresponsive Nanoporous Membranes by Coating Anodized Alumina with Poly(3,4ethylenedioxythiophene) and Polypyrrole. 2014, 299, 190-197.

Nerve modelisation

As seen in the other parts of this wiki, we chose to use an nanoporous membrane in our device. The first goal of the membrane was to confine our biofilm, so it does not escape the prosthesis. Moreover, we also used our membrane as a conductive electrode. This solution was interesting since we didn’t have enough time to develop an entire electrical device which collect and treat the signal of the nerves. However, we know we still need to improve our interface if we want the patient to fully control his prosthesis. That is why we decided to look at what is already made in this field. So, first, we detailed how it is possible to model the electrical characteristics of a nerve. Then, we searched information on electrodes and signal treatment.

The more selective an electrode is the simpler the extraction of information. Thus, the maximum selectivity, being reduced to the activity of a single fiber, is required for the measurement interfaces. Unfortunately, this search for selectivity will lead to a search for proximity between the electrode and the fibers, at the detriment of the nerve’s physical integrity. Indeed, the risk of infection or trauma to the body increases with the invasiveness of the electrodes. Electrodes can therefore be classified according to criteria such as selectivity and invasiveness. The ideal electrode is one that has the highest selectivity while remaining the least invasive possible. To make a choice, a compromise must be made between the selectivity and the degree of invasiveness of the electrode. The "secondary" criteria are stability and repeatability. We will present the neural electrodes by exposing their performances in terms of selectivity and level of invasiveness.

I. Extra-neural electrodes

1. Helicoidal electrode interface:

Helicoidal electrodes are placed surrounding the nerve and are made of flexible metal ribbon in a helical design. This design allows the electrode to conform to the size and shape of the nerve to minimize mechanical trauma. The structural design causes low selectivity. Helicoidal electrodes are currently used for functional electrical stimulation, to control intractable epilepsy, sleep apnea, and to treat depressive syndromes.

2. Cuff electrode:

Considered as extraneural electrodes, cuff electrodes are widely used to perform basic and applied electro-neurophysiology studies and are particularly interesting for their ability to achieve good nerve recruitment with low thresholds. The cuff-style electrode provides a cylindrical electrode contact with a nerve for each of an arbitrary number of contacts, is easy to place and remove in an acute nerve preparation, and is designed to fit on the nerve (Cf. Figure 1). For each electrode, the electrical contacts were cut from metal foil as an array so as to maintain their positions relative to each other within the cuff. Lead wires were soldered to each intended contact. The structure was then molded in silicone elastomer, and individual contacts were electrically isolated. The final electrode is curved into a cylindrical shape with an inner diameter corresponding to that of the intended target nerve. These electrodes have been successfully used for nerve stimulation, recording, and conduction block in a number of different acute animal experiments by several investigators.

Figure 1: Schematic of a nerve cuff electrode. Retrieved on Oct. 14th from MicroProbes for Life Science[1]

The activity recorded by the cuff electrode represents the simultaneous activity of a large number of active axons. The potential of action seen by the electrode are overlapped, allowing only a "global" image of the activity inside the nerve. As a result, the selectivity of the recording is limited by the number of axons undergoing simultaneous discharge and by the position and surface of the contact of the cuff electrode. This type of measurement therefore does not allow the identification of fiber activity alone.

Increasing the number of electrode poles allows to increase the selectivity of this type of electrode. A multi-pole cuff electrode is then called a cuff electrode having more than three contacts. These contacts can be rings or segments of rings.

3. FINE electrode:

The flat-interface nerve electrode (FINE) was designed for selective nerve recording by realigning the fascicles and reshaping the nerve into a more flattened cross section which increases the surface area of the exposed nerve and offers greater access to fascicles. This kind of electrode is particularly interesting as it was possible to achieve more than 90% selectivity (Cf. Figure 2)

Figure 2: Cross section and schematic of a FINE electrode[2]

II. Information extraction:

1. Extraction of the discharge frequency:

The most relevant information to extract is the discharge frequency of active fibers because it represents the means of coding information by the nervous system. Significantly, such processing must be applied to signals representing the activity of a limited number of fibers. In fact, the published examples relate exclusively to intra-neural collection: the only method, today, which allows to observe the activity of fibers alone. However, since we don’t want to use intra-neural electrode in our device we will not detail how to extract the discharge frequency.

2. Envelope extraction:

Rectification and Bin-Integration (RBI) of the nerve raw signal is widely used in rehabilitation application. This point of RBI ENG are found by calculating the average of the absolute value of ENG samples spread over a given period of time. This period is called “bin” and its value depends on the application. It ranges from 10 ms to 200 ms. The smoothed envelope-like signal created by RBI makes it easy to extract information about the innervated organ.

III. Improvement of the electroneurogram records selectivity

1. ENG-EMG selectivity:

2. Type of nerve fiber selectivity:

3. Spatial selectivity:

IV. An example of the development of a multi-channel acquisition device

References