Supplementary Information for Organic electronics for ... · Supplementary Discussion Design and Function of the Delivery Device. ... (MicroChem), leaving openings for the liquid

SUPPLEMENTARY INFORMATIONdoi: 10.1038/nmat2494

nature materials | www.nature.com/naturematerials 1

Supplementary Information for

Organic electronics for precise delivery of

neurotransmitters to modulate mammalian sensory

function

by D.T. Simon, et al.

Supplementary Discussion

Design and Function of the Delivery Device. The planar delivery devices employed the same

geometrical design as previously reported18

. The device comprised three regions of

photolithographically patterned PEDOT:PSS referred to as A, C, A’, and C’ for anode, cathode, and

anode and cathode supplemental, respectively (Supplementary Figure 1). A’ and C’ were used to

increased the electrochemical capacity of the anodic and cathodic systems, respectively – i.e., they

provided additional PEDOT to undergo redox reactions. The A and C electrodes, initially formed

from a single region of PEDOT:PSS, were isolated by over-oxidizing the PEDOT connecting

them36,37

, thus disabling electronic conductivity while maintaining ionic conductivity. The devices

were then encapsulated with hydrophobic SU-8 2010 photoresist (MicroChem), leaving openings for

the liquid to be applied over the electrodes. Finally, electrical connection was made as shown in

Figure 1 and Supplementary Figure 2. Upon application of a voltage, the anodic (A-A’) and cathodic

(C-C’) systems undergo the electrochemical reactions of Eqns. 1 and 2 above, i.e., ion release from A

and uptake into C, where the (water-soaked) over-oxidized region connecting A and C acts as an

exchange layer providing selective transport of cations. This is due to the fact that the polyanionic

PSS allows significant transport of cations only. The encapsulated device operates via a similar

principle except that the target system (Figure 1d) constitutes the salt bridge between anodic and

cathodic systems.

An interesting feature of both device geometries is that voltages in excess of 30 V can be

applied to the PEDOT:PSS electrodes with negligible resulting electric fields in the target system.

This is due to the significantly higher electronic conductivity of the electrodes compared to the ion

channel (composed of over-oxidized PEDOT:PSS), i.e., the voltage drop occurs primarily across the

channel. Even in the case of the encapsulated device, where the target system can be considered to be

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SUPPLEMENTARY INFORMATION doi: 10.1038/nmat2494

halfway through the ion channel (cf. Figure 1), consideration of the device geometry shows that only a

small fraction of the voltage would be dropped within the target electrolyte. Indeed, control

experiments where only voltage was applied (as described in Methods) yielded null effect.

An additional advantage of utilizing PEDOT:PSS is that, in comparison to the metals commonly

found in electrophoretic devices, problematic secondary electrochemical reactions can be avoided.

For metal-solution interfaces, the redox reactions take place in solution, often leading to gas formation

or potentially toxic side products – any of which would be detrimental to a delivery device’s viability.

For PEDOT:PSS and other conducting polymer electrodes, the redox reactions can take place within

the polymer. Thus, even at higher voltages where gas evolution or hydrolysis would be expected for

metal electrodes, the electrodes in the device described in this manuscript simply undergo the

PEDOT

0 PEDOT

+ reaction.

The device operates in the electrochemical fashion described in the text and above so long as

there is available PEDOT0 in the anode (source electrode) for oxidation and PEDOT

+ in the cathode

(target electrode) for reduction. Thus, the functional lifetime is limited by the volume of PEDOT:PSS

in each electrode, i.e., the electrode area. This can of course be tailored by simply adjusting the

geometry of the electrodes to provide more or less starting material as needed. The functional lifetime

could also be effected by operating the device in a pulsed fashion, allowing recovery of redox sites

between delivery periods, e.g., by reversing the bias voltage, or possibly allowing the device to remain

off for a given amount of time.

The planar device has previously been shown to be biocompatible. A variety of cell types, such

as neurons, epithelial and endothelial cells, fibroblasts, macrophage-like cells, and T cells, were

cultivated on the PEDOT:PSS electrodes of planar devices. Analysis of adhesion, morphology, and

viability indicated that the device exhibited similar biocompatibility for all cell types as compared to

standard substrates18

. In the case of the encapsulated device, the additional materials used during

fabrication (e.g., surgical-grade tubing, silicone encapsulant) each have a precedent in biomedical

research. Furthermore, as seen in Figure 4a, only the tip of the device is in contact with the RWM,

meaning that none of these additional materials are in direct contact with the tissue involved in the

experiment. Given the acute nature of the in vivo experiment, the encapsulated device can thus be

deemed biocompatible for this application.



Calculation of Device Efficiency. The efficiency of the device is determined by comparing the

number of electrons passed through the driving circuitry (determined by integrating the current

measured by the Keithley 2602 SourceMeter) to the number of intended ions delivered to the target

system (determined by quantitative HPLC analysis of the target solution, described in Methods). This

ratio can be determined by fitting the delivered concentration, [M], vs. integrated charge, Q, data with

a straight line with non-zero Q offset. The results of this analysis are shown in Supplementary Figure

3. The non-zero Q offset corresponds to the transport of other ions in the PEDOT:PSS film, present

from original manufacture or from the photolithographic processing, before the arrival of the intended

ions. In other words, any ions present in the film will be electrophoretically transported. Only after

the “front” of intended ions from the source solution makes its way across the channel will a rise in

concentration be observed. Once the data is fit, the electron:molecule ratio, R, can be calculated as:

R ≡ m ⋅V ⋅e ⋅NA( )

−1

,

where m is the slope of the [M] vs. Q fit; V is the total volume of the target solution; e is the

elementary charge, 1.602×10-19

C; and NA is Avogadro’s number, 6.022×1023

mol-1

. The results of the

ratio calculation are shown in Supplementary Figure 3. For materials with low pKa (Glu pKa: 2.13;

Asp pKa: 1.99; GABA pKa: 4.03)38

, the excess protons present in the source solution will also be

pumped. Owing to the smaller size of protons compared to Glu or Asp, their mobility through the

channel can be significantly higher than the intended materials. This is the reason for the significantly

higher electron:molecule ratios for Glu and Asp.

Supplementary Methods

Fabrication of Planar Devices. PEDOT:PSS coated polyethylene terephthalate (PET) substrate

(commercially available as Orgacon™ EL-350 from AGFA-Gevaert), was cleaned with acetone and

deionised water, then dried in an oven at 110 ºC. The layer of PEDOT:PSS was patterned into the

separate electrodes and ion channel by etching the regions not covered with Shipley 1818 photoresist



(MicroChem) with an O2/CF4 plasma. After removal of the photoresist, another layer of Shipley 1818

was deposited and patterned to provide an opening to the ion channel region of the PEDOT:PSS. The

substrate was then dipped into to 1% v/v NaOCl(aq) followed by a deionised water rinse, thereby

over-oxidizing the ion channel, i.e., the region not covered by the second layer of photoresist. This

second layer of photoresist was then removed, and a layer of SU-8 2010 photoresist was deposited and

patterned as the top insulating layer, thus defining the openings for the electrolytes. Conductive paint

was applied to facilitate electrical contact to the electrode pads. All photolithographic patterning was

carried out using a Süss MA6/BA6 mask and bond aligner (Süss MicroTec). Approximately 24 hr

before use, the devices were soaked in deionised water, saturating the PEDOT:PSS and enhancing its

ionic conductivity.

Fabrication of Encapsulated Devices. Similar to the fabrication of planar devices, the

PEDOT:PSS coated polyethylene terephthalate (PET) substrate (commercially available as Orgacon™

EL-350 from AGFA-Gevaert), was cleaned with acetone and deionised water, then dried in an oven at

110 ºC. The substrate was then exposed to an O2 plasma to pre-treat the surface for photoresist

adhesion. The layer of PEDOT:PSS was patterned into the separate electrodes and ion channels by

etching the regions not covered with Shipley 1818 photoresist (MicroChem) with an O2/CF4 plasma.

After removal of the photoresist, the channel regions were masked with adhesive tape and an

additional layer of PEDOT:PSS was applied by bar-coating using a solution of 95% w/w ICP-1010

PEDOT:PSS solution (AGFA- Gevaert), 5% w/w diethylene glycol, 0.1% w/w Zonyl FS-500

fluorosurfactant (Fluka), <0.1% w/w Silquest A-187 silane (OSi Specialties) solution and a PA-2105

Automatic Film Applicator (BYK-Gardner) bar-coater. After baking at 100 ºC for 10 min and removal

of the masking tape, the substrate was cut into two-electrode pieces using a computer-controlled

Graphtec FC2200 Cutting Plotter table. Each two-electrode piece was then fitted with two 3 cm

lengths of Silastic silicone tubing (Dow Corning). The end of the tubes nearest the ion channels was

sealed with Sylgard 186 silicone encapsulant (Dow Corning) and cured at 110 ºC for 35 min. Each

device was then dipped into 1% v/v NaOCl(aq) and rinsed in deionised water, thus over-oxidizing the

ion channels. After drying, a thin layer of Sylgard 184 or 186 (Dow Corning) was applied over the ion

channels leaving only the last ~1 mm exposed (the dotted region of Figure 1d), and conductive paint

was applied on the other end of the device (far left of Figure 1d) to provide electrical contact. The



devices were cured at 110 ºC for 35 min, then stored in air. Approximately 24 hr before use, the

devices were soaked in deionised water, saturating the PEDOT:PSS. Just before use, the tubes were

blown out with compressed air and filled with the solutions using a syringe. The open ends of the

tubes were sealed with either a tube clamp or Xantopren L/Blue dental glue (Bayer).

Electrical Control and Monitoring of the Devices. All the devices were controlled using a

Keithley 2602 SourceMeter (Keithley Instruments) controlled via a custom designed LabVIEW

interface. Devices were either operated in constant voltage mode, where a dc voltage was applied

between the device’s two electrodes, or in constant current mode, where a dc current was applied. In

both cases, the current and voltage across the device were recorded at specified time intervals (usually

1 s). The time integrated current passing through the device was used to determine the theoretical

maximum amount of ions pumped. Devices were connected in the schemes of Figure 1a or 1d.

Material Quantification Using High Throughput Liquid Chromatography (HPLC).

Solutions of 0.1 M L-glutamic acid(aq) (Sigma), L-aspartic acid(aq) (Sigma), or GABA(aq) (Tocris),

in 150 µl volumes, were placed over the source electrode while phosphate buffer solution (PBS) was

used as target electrolyte. The Glu solution was reduced in pH by addition of HCl to promote the

cationic form of the substance. Asp and GABA were dissolved directly in deionised water. Devices

were operated by applying a dc voltage of 4 or 8 V between the source and target sides, and the

current was simultaneously monitored. After 200, 400, 600, 800, and 1000 s, the target solution was

removed and subsequently measured by HPLC with fluorescence detection. Briefly, 10 µl of the

solutions were mixed with 10 µl of an o-phthaldialdehyde reagent for 1 min at 4 ºC with a CMA/200-

240 refrigerated microdialysis sampler and sample injector (CMA/Microdialysis AB, Stockholm,

Sweden). 15 µl of the mixed solution was subsequently injected on a 100×4.6 mm Chromolith

performance RP-18e HPLC column (Merck KGaA, Darmstadt, Germany). The column was eluted

with a solution of sodium acetate, methanol and tetrahydrofurane at pH 6.9 with a gradient pump

(Spectra Physic SP8800). A gradient of methanol was used to clean and regenerate the column. The

derivatives of the amino acids were detected with a CMA/280 fluorescence detector at an excitation

wavelength band of 340-360 nm and an emission wavelength band around 495 nm.



Cell Culturing. Primary mouse cortical cultures were prepared essentially as previously described32

.

Briefly, cerebral cortices from postnatal day 4-5 mice were dissected clean of meninges, hippocampi

and thalaminc regions before being mechanically and chemically dissociated. Cells were plated and

maintained in minimum essential medium (MEM) with 10% fetal bovine serum (FBS) for 5-7 days

after which neurons and microglia were removed by shaking. The cells were lifted using trypsin-

EDTA, replated, and used for experiments after 4-5 days.

Intracellular Calcium Imaging. Cells were grown in cell culture dishes and incubated with the

membrane-permeable Ca2+

-sensitive dye FURA-2 AM (2 µM, Molecular Probes) and 0.02% pluronic

acid (Molecular Probes) in 37 °C for 1 h. The dye was removed and replaced with fresh cell media.

The delivery device was prepared as previously described using 0.1 M L-glutamic acid(aq) as the

source electrolyte and 0.1 M NaCl(aq) as the cathodic electrolyte. Cells were monitored using an

upright Nikon Eclipse 80i with a 40 x /0.80 epifluorescence objective. Excitation at 340 and 380 nm

was achieved with a DeltaRAM illuminator and a DeltaRAM-V monochromator with a computer

controlled SC500 shutter controller. Emission (510 nm) was collected every 10 s with a Photometrics

Coolsnap CCD camera. Data were analyzed using PTI ImageMaster3 Software. The experiment was

repeated three times and representative recordings from transported Glu are presented.

Guinea Pigs, Surgical Procedure, and Control Experiments. Hartley strain guinea pigs

weighing 250–400 g without any evidence of middle ear pathology were used for this study. The

Ethical Committee at the Karolinska Institutet approved the care and use of animals in this experiment.

Animals were anesthetized with an intramuscular injection of 50 mg ketamine and 8 mg xylazine per

kg body weight. The otic bulla of the temporal bone of a guinea pig was exposed using a retroauricular

approach. A small hole was made on the bulla to expose the round window membrane. The ion pump,

which was loaded with 0.1 M L-glutamic acid in 10 µM HCl(aq) (pH ~3) as the source electrolyte and

0.1 M NaCl(aq) as the cathodic electrolyte, was mounted with the delivery channel’s outlet in direct

contact with the RWM using the physiological fluid present on the surface of the RWM as the target



electrolyte. To eliminate the possibility that the protons delivered along with the Glu from the pH ~3

source solution effected the results, control experiments were performed using 0.1 M HCl(aq) (pH 1)

as the source solution. Other control experiments included delivery of Na+ ions from a 0.1 M NaCl(aq)

source solution to assure that the electric field at the tip of the device was not altering auditory

sensitivity and sham operations to control for the surgical procedure.

Monitoring Auditory Function. Auditory function was assessed by ABR recordings to detect in

real time any excitotoxic effect. Measurements of ABR thresholds were performed at frequencies of 8,

16 and 20 kHz before Glu delivery and 0, 15, 30 and 60 min after Glu delivery. ABR thresholds were

recorded with subcutaneous stainless steel electrodes as the potential difference between an electrode

on the vertex and an electrode on the mastoid, while the lower back served as ground. The stimuli

were generated through Tucker-Davis Technologies (Gainesville, FL, USA) equipment controlled by

computer. The acoustic stimuli, consisting of tone pip stimuli, was delivered through ES1 speakers

(Tucker-Davis Technologies) for open-field stimulation 8 cm away from the ear and presented at a

repetition rate of 20 Hz. The evoked responses were amplified 100000 times and averaged from 2-3

repeated measurements. The stimuli were presented well above threshold and decreased in 5 dB steps

until threshold was found. Threshold was defined as the lowest intensity at which a visible ABR wave

was seen in two averaged runs. The cochleae were collected for histological analysis and fixed in 4%

PFA in 1% PBS at 4 °C for 2 h.

Histological Analysis. After decalcification with the rapid decalcifying agent RDO (Apex

Engineering Products Corporation, IL, USA) for 2 h at room temperature, 10 µm thick cryostat

sections were prepared. Sections were stained with cresyl violet. Between 20-25 sections from each

specimen was viewed with Zeiss Axioskop microscope and 100x oil objective.

Statistical Analysis of ABR Results. The overall effects of Glu delivery on ABR threshold

shifts as a function of time (Figure 4c) for the different frequencies were examined using two-way

factorial analysis of variance. When the interactions were significant (p < 0.05), multiple comparisons



using the Fisher protected least significant difference test were performed for pairwise comparisons.

Analysis was performed between the Glu response (n = 5) at 15 min and 60 min at each frequency,

yielding p < 0.0001, p = 0.0012, and p = 0.0002 for 20, 16, and 8 kHz, respectively. These p values are

indicated by the asterisks to the left of the 60 min Glu bars in Figure 4c. Analysis was also performed

between the Glu and H+ (n = 3) responses at 60 min at each frequency, yielding p < 0.0001, p =

0.0025, and p < 0.0001 for 20, 16, and 8 kHz, respectively. These p values are indicated by the

asterisks to the right of the 60 min Glu bars (Figure 4c).



Supplementary Figures and Legends

Supplementary Figure 1 Histological section near the apex of the cochlea after Glu delivery for 60

min. The inner hair cells can be seen on the right and the outer hair cells on the left. The section

indicates no excitotoxic effect to the dendrites below the inner hair cells (as seen in Figure 4d). Scale

bar: 20 µm.

Supplementary Figure 2 Planar device geometry. a, Top view and connection scheme for the

planar device with geometry to scale. The device is 25 mm in diameter. b, Cross section through the

A and C electrodes. The electrode reactions and schematic ion transport are listed below the

associated electrodes, while the layered structure is explained in Figure 1. In both parts of the figure,

the dotted layer indicates the hydrophobic encapsulation and the horizontally hashed region joining A

and C is the over-oxidized channel.



Supplementary Figure 3 Determination of device efficiency. Results for a, Glu, b, Asp, and c, GABA

in the planar geometry, and d, Glu in the encapsulated device. The target solution volumes were

either 150 µl or 100 µl for the planar or encapsulated geometries, respectively. The fits are straight

lines with non-zero Q offset, and the calculated electron:molecule ratios, R, are reported.



Supplementary Notes

Supplementary reference list. Numbering continues from the reference list in main manuscript.

36 Krische, B. and Zagorska, M., Overoxidation in conducting polymers. Synthetic Metals 28, 257-

262 (1989).

37 Tehrani, P. et al., Patterning polythiophene films using electrochemical over-oxidation. Smart

Materials and Structures 14, N21-N25 (2005).

38 Dissociation Constants of Organic Acids and Bases, in CRC Handbook of Chemistry and Physics,

88th Edition (Internet Version 2008) (ed. Lide, D. R.) 8.42-8.51 (CRC Press/Taylor and Francis,

Boca Raton, FL, 2008).

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Supplementary Information for Organic electronics for ... · Supplementary Discussion Design and Function of the Delivery Device. ... (MicroChem), leaving openings for the liquid