Insertion of Flexible Neural Probes Using Rigid Stiffeners Attached with Biodissolvable Adhesive

Podsumowanie

Insertion of flexible neural microelectrode probes is enabled by attaching probes to rigid stiffeners with polyethylene glycol (PEG). A unique assembly process ensures uniform and repeatable attachment. After insertion into tissue, the PEG dissolves and the stiffener is extracted. An in vitro test method evaluates the technique in agarose gel.

Streszczenie

Microelectrode arrays for neural interface devices that are made of biocompatible thin-film polymer are expected to have extended functional lifetime because the flexible material may minimize adverse tissue response caused by micromotion. However, their flexibility prevents them from being accurately inserted into neural tissue. This article demonstrates a method to temporarily attach a flexible microelectrode probe to a rigid stiffener using biodissolvable polyethylene glycol (PEG) to facilitate precise, surgical insertion of the probe. A unique stiffener design allows for uniform distribution of the PEG adhesive along the length of the probe. Flip-chip bonding, a common tool used in microelectronics packaging, enables accurate and repeatable alignment and attachment of the probe to the stiffener. The probe and stiffener are surgically implanted together, then the PEG is allowed to dissolve so that the stiffener can be extracted leaving the probe in place. Finally, an in vitro test method is used to evaluate stiffener extraction in an agarose gel model of brain tissue. This approach to implantation has proven particularly advantageous for longer flexible probes (>3 mm). It also provides a feasible method to implant dual-sided flexible probes. To date, the technique has been used to obtain various in vivo recording data from the rat cortex.

Wprowadzenie

Microelectrode arrays are an essential tool in neuroscience as well as emerging clinical applications such as prosthetics. In particular, penetrating micro-electrode probes enable stimulation and recording of neuronal activity through close contact with cells in the brain, spinal cord, and peripheral nerves. A major challenge for implanted neural probes is stability and longevity of the stimulation and recording functions. Modeling and experimental studies of the interaction between microelectrode probes and neural tissue have suggested that one mechanism for degradation is micro-tearing of neural tissue due to slight relative motion between the probe and tissue ^1-3. One solution is to fabricate flexible probes that match more closely the bulk stiffness properties of neural tissue in order to minimize relative micromotion. As such, biocompatible thin film polymers such as polyimide and parylene have been adopted as favorable substrates for microelectrode probes ^4-8.

A tradeoff of flexible probes is that they are difficult to insert into the neural tissue. Researchers have taken various approaches to facilitate insertion of flexible probes while preserving the desirable mechanical properties. One class of designs modifies the polymer probe geometry to increase stiffness in certain sections or axes while maintaining compliance in other parts. This has been accomplished by incorporating ribs or layers of other materials ^9,10. Another approach integrates a 3-D channel into the polymer probe design that is filled with biodegradable material ¹¹. This probe can be temporarily stiffened, and after insertion the material in the channel dissolves and drains out. However, methods such as these that permanently modify the geometry of the final implanted device may compromise some of the desirable features of the flexible probe.

One method that does not alter the final probe geometry is to encapsulate the polymer device with biodegradable material to temporarily stiffen the device ^12-14. However, typical biodegradable materials have Young's moduli orders of magnitude smaller than that of silicon and would consequently require larger thickness to achieve the same stiffness. Adequately coating the probe can result in a more rounded or blunt tip, making insertion more difficult. Also, since dissolvable coatings are exposed, there is a risk of them dissolving immediately upon contact, or even close proximity, with the tissue.

Another class of methods uses novel probe substrate materials that reduce in stiffness after being implanted into tissue. Such materials include shape memory polymers ¹⁵ and a mechanically adaptive nanocomposite ¹⁶. These materials are able to decrease in elastic modulus significantly after insertion, and can result in probes that more closely match the mechanical properties of neural tissue. However, the achievable range of stiffness is still limited, so they may not be able to provide very high stiffness equivalent to silicon or tungsten wires. Thus in the case of flexible probes that are very long (e.g. >3 mm) or that have extremely low stiffness, a method of temporarily attaching a more rigid stiffener may still be required.

Yet another promising method reported is to coat a stiffening shuttle with a permanent self-assembling monolayer (SAM) to customize the surface interaction between the shuttle and the flexible probe ¹⁷. When dry, the probe adheres to the coated shuttle electrostatically. After insertion, water migrates onto the hydrophilic surface, separating the probe from the shuttle so that the shuttle can be extracted. Shuttle extraction with reduced probe displacement was demonstrated (85 μm). However, with only electrostatic interactions holding the probe to the shuttle, there is some risk of probe slippage relative to the shuttle before and during insertion.

We have developed a method in which the flexible probe is attached to a stiffener with a temporary biodissolvable adhesive material that securely holds the probe during insertion. The probes used were made of polyimide, which has an elastic modulus on the order of 2-4 GPa. The stiffener was fabricated from silicon, with an elastic modulus of ~200 GPa. When attached, the stiffness of the silicon dominates, facilitating insertion. Once inserted into the tissue, the adhesive material dissolves and the stiffener is extracted to restore the probe to its initial flexibility. We selected polyethylene glycol (PEG) as the biodissolvable adhesive material. PEG has been used in implanted applications such as neural probes, tissue engineering, and drug delivery ^11,18,19. Some evidence has suggested that PEG may attenuate neuroinflammatory response in brain tissue ^18,20. Compared to other possible materials, including sucrose, poly lactic-co-glycolic acid (PLGA), and polyvinyl alcohol (PVA), PEG has a dissolution time in biological fluids that is of an appropriate scale for many implant surgeries (on the order of tens of minutes, depending on molecular weight). In addition, it is solid at room temperature and liquid at temperatures ranging from 50-65 °C. This property makes it particularly suitable for our precision assembly process. Moreover, similar to the SAM described in ¹⁷, the dissolved PEG is hydrophilic, facilitating extraction of the stiffener. This advantageous approach is enabled by a novel stiffener design and methodical assembly process which ensure uniform adhesive coverage and accurate and repeatable alignment. In addition to the assembly process, we present the method of implementing the removable stiffener during surgery, as well as an in vitro procedure to evaluate extraction of the stiffener.

The protocol presented herein assumes that the user possesses a flexible polymer microelectrode probe. The part of the protocol relating the fabrication of the stiffener and assembly of this probe to a stiffener assumes access to common tools found in a microfabrication facility. The protocol relating to insertion and extraction would likely be performed in a neuroscience-oriented laboratory.

Protokół

1. Assembly of Probe to Stiffener

This section of the protocol describes fabrication of a silicon stiffener, and the assembly of a thin-film polymer probe to the stiffener. Figure 1 illustrates a typical polymer neural probe along with the proposed stiffener. The details of the stiffener design are shown in Figure 2. The novel feature of this design is the shallow "wicking" channel running along its length which is used to distribute liquid adhesive during assembly. The wider portion of the stiffener is a tab for handling during assembly and surgical insertion. A reservoir on the tab connects to the channel. The component is fabricated from silicon using standard microfabrication processes.

1. The silicon stiffener with a wicking channel was fabricated from a silicon-on-insulator (SOI) wafer with a device layer thickness equal to the desired thickness of the stiffener (Figure 3A). A reasonable range of stiffener thickness is 20-100 μm. It is recommended that the width of the stiffener be 20-30 μm smaller than the probe width, which helps to prevent overflow of the adhesive from the bond interface to the top of the probe. First the wicking channels are dry-etched using the standard Bosch process (Figure 3B). Next, the stiffener geometry is defined by a longer etch that stops on the buried oxide layer (Figure 3C). Finally, the stiffeners are released by wet-etching the buried oxide layer in 49% hydrofluoric acid (Figure 3D). After thoroughly rinsing the stiffeners, soak them in deionized water for 15 min.
2. Place a pellet of polyethylene glycol (PEG) of molecular weight 10,000 g/mol into the reservoir (Figure 4). Heat the stiffener to 65 °C so that the PEG melts and wicks into the channel by capillary action. Then cool to room temperature to solidify.
3. Figure 5 shows a schematic of the flip chip bonder set up. Place the stiffener upside down on the base stage of the flip chip bonder, then pick up the stiffener with the tool head. Place the probe upside down on the base stage. Using the flip chip bonder, align the stiffener and the probe and then lower the stiffener and place it onto the probe.
4. The base stage of the flip chip bonder should have a heating element to apply heat to the substrate. After placing the stiffener, heat the assembly once again to 65 °C. Allow one minute for the PEG to remelt and begin to fill in the interface between the probe and stiffener. Cool to solidify.
5. Turn the assembly over and inspect from the top. Reheat as needed to allow the PEG to completely fill the interface between the probe and the stiffener. This can be visually evaluated since the probe is transparent. As the assembly is sitting on the heater top- (probe-) side up, manually place 1-3 extra pellets of solid PEG onto the tab so that they melt over the probe, providing additional reinforcement in this region (Figure 6). Finally, allow the assembly to cool so that the PEG solidifies. At this point, the assembly is ready for surgical insertion.

2. Insertion and Extraction

1. Mount the probe-stiffener assembly to a micromanipulator as illustrated in Figure 7A by adhering the back of the stiffener to the micromanipulator arm at the tab region. This may be done with double-sided tape or cement, but take care not to contact the probe with adhesive. Temporarily secure the connector end of the probe to the micromanipulator with a small piece of adhesive putty such that it can be easily removed with low force.
2. Position the probe assembly over the target and insert the probe with the desired insertion speed. Insertion speeds of 0.13-0.5 mm/sec were used when developing this protocol.
3. Immediately remove the connector end of the probe from the micromanipulator gently and rest it on a nearby surface, such as a second manipulator arm (Figure 7B). This must be done before the PEG begins to dissolve to avoid displacing the probe.
4. Allow time for PEG to dissolve. This amount of time will depend on PEG molecular weight and area of contact between the probe and stiffener. For example, with PEG molecular weight of 10,000 g/mol, a microelectrode probe about 6 mm and a matching stiffener that is 306 μm wide, 15 min has been found to be an adequate amount of time. Section 3 of the protocol presents a method to test the required dissolution time. During this time, apply phosphate buffered saline (PBS) using a dropper around the tab and insertion point to dissolve any PEG that is above the target (Figure 7C).
5. Using a motorized micropositioner, begin extraction of the stiffener by applying a displacement of 100 μm at a speed of 5 mm/sec. This initial fast motion helps to overcome any static friction and minimize probe displacement. Then, complete the stiffener extraction at a slower speed of approximately 0.1 mm/second (Figure 7D).
6. In the case of an actual surgery, continue with normal procedures to apply gel, silicone, and/or dental acrylic at the insertion site to secure and protect the probe, as demonstrated in ²¹.

3. Agarose Gel Test

This section of the protocol describes a set up and procedure to examine the extraction of the stiffener in a 0.6% agarose gel that approximates the bulk mechanical properties, pH, and salinity of brain tissue ^17,22. Since the gel is nearly transparent through short distances, stiffener separation and probe displacement can be observed.

1. Prepare a solution of 0.6% agarose in phosphate buffered saline (PBS). Mix at an elevated temperature to completely dissolve the agarose powder. Pour the solution into a shallow acrylic box; gel should be 3/4- 1 in deep. Allow to the gel set at room temperature for an hour.
2. Ensure that the hardened gel is saturated with PBS so that it does not dry out, and heat the gel to 37 °C.
3. Set up the micromanipulator, box of agarose gel, and microscopic camera system as shown in Figure 8.
4. Insert a glass reference fiducial into the box of gel by sliding it between the gel and the side of the box (Figure 8). Use a dental pick to square the features on the reference fiducial to the field of view of the digital microscope.
5. Mount the probe assembly to the micromanipulator as described in step 2.1.
6. Position the probe assembly over the gel about 1 mm behind the reference fiducial.
7. Insert the probe into the gel, using the camera to guide it to a desired depth in the field of view.
8. Immediately move the connector end of the probe to rest on a nearby surface.
9. Make any required adjustments to the camera image to focus on the probe (the reference fiducial features may be slightly out of focus). Take a snapshot of the probe location.
10. Allow PEG to dissolve (this time may vary, and in fact may be a parameter to be tested). Apply PBS near the tab to dissolve PEG that is above the gel.
11. Start video capture if desired, and begin extraction of the stiffener as described in step 2.5. When extraction is complete, take a final snapshot of probe location.
12. Use image processing tools to compare the images before and after stiffener extraction. Use the features on the reference fiducial that are visible in the field of view to register (align) the images. Calibrate the scale of the image based on the size of known features on the probe. Measure the distance of probe displacement.

Wyniki

This insertion technique was used in conjunction with LLNL thin-film polyimide probes, which have passed ISO 10993 biocompatibility standards and are intended for chronic implantation. A typical thin-film polyimide probe is illustrated in Figure 1 along with a silicon stiffener that is approximately 10 mm long in the narrow region. This stiffener has one wicking channel running along its length, as shown in Figure 2. Figure 3 illustrates the mimcrofabrication process u...

Dyskusje

The method described here provides a well-controlled process to attach thin-film polymer probes to separate stiffeners with a biodissolvable adhesive. Also presented is the recommended surgical procedure to implement these removable stiffeners and a technique to validate the procedure in vitro for a given probe-stiffener configuration. Since the stiffener can be made arbitrarily rigid, the method can facilitate insertion of relatively long probes (>3 mm). As such, the insertion method is expected to be an e...

Materiały

Name	Company	Catalog Number	Comments
Polyethylene glycol, 10,000 g/mol	Sigma Aldrich	309028
Agarose	Sigma Aldrich	A9539
Flexible Sub-micron Die Bonder	Finetech	Fineplacer lambda
Micromanipulator	KOPF	1760-61
Digital Microscope	Hirox	KH-7700
Dual Illumination Revolver Zoom Lens	Hirox	MXG-2500REZ
Precision Motorized Actuator	Newport	LTA-HS	w/ CONEX-CC controller