Proton conductivity in ampullae of Lorenzini jelly

We collected samples by squeezing AoL jelly from visible surface pores on the skin of bonnethead shark (Sphyrna tiburo), longnose skate (Raja rhina), and big skate (Raja binoculata) (Fig. 1, A and B, and fig. S1). After storing at −20°C, we deposited the AoL jelly directly onto the sample surface without further processing. For all species, the resulting AoL jelly is thick, viscous, and optically clear (Fig. 1E). We exposed AoL jelly samples to 90% relative humidity (RH) for 30 min and hydrated them to 97% water content as measured by thermogravimetric analysis (TGA) (fig. S2). This water content is consistent with previous reports (10, 11). If allowed to dry in air, the volume of the sample decreases by approximately 80%, corroborating the known fact that the jelly is a hydrogel with a high water content.

We performed initial electrical measurements in a standard two-terminal geometry with palladium (Pd) source and drain contacts (Fig. 2A) (12–15). When Pd is exposed to hydrogen, Pd forms palladium hydride (PdH_x) with a stoichiometric ratio x ≤ 0.6. With a source-drain potential difference, V_SD, the PdH_x source and drain inject and drain protons (H⁺) into and from the AoL jelly samples, effectively serving as protodes (12–15). For each H⁺ injected into the AoL jelly, an excess electron is collected by the leads, which complete the circuit and result in a current measured at the drain, I_D (Fig. 2A). For an applied V_SD = 1 V, we measured I_D at room temperature as a function of time (t) in an atmosphere of nitrogen or hydrogen with controlled RH, as we have previously done for the biopolymer melanin (16). For RH = 50%, the AoL jelly samples extracted from R. rhina have very low electrical conductivity with I_D ~ 3 nA when measured with electrically conducting and proton-blocking Pd contacts exposed to nitrogen (Fig. 2B, black curve). At low RH, the only contribution to I_D is likely from electrons, because ions and protons require a highly hydrated material to conduct (17). For RH = 75%, I_D increases with a 10-nA peak when measured with Pd contacts (Fig. 2B, blue curve). The formation of a Debye layer from the ions blocked at the contact–AoL jelly interface causes I_D to rapidly decrease to 0.3 nA at t = 20 s. Because Pd contacts block ions and protons, I_D = 10 nA is likely the ionic component to the transient current in the AoL jelly, which contains the same ionic species as seawater (table S1) (11). The observed capacitance (22 nF) is consistent with the estimated capacitance for ionic charging of the Pd–AoL jelly interface (26 nF) (table S1). We observed a similar behavior for ionic currents in the biopolymer melanin (16). This ionic component corresponds to an ionic conductivity of the AoL jelly, σ_ion = 100 nS/cm. This σ_ion is rather low compared to the conductivity of seawater (≈40 mS/cm), which was also attributed to the AoL jelly by previous work (11). However, larger ions such as Na⁺ and Cl⁻ require larger pores in the material to be able to diffuse, and low ionic conductivity of the AoL jelly was also previously observed (18). For proton-conducting PdH_x contacts at 75% RH, I_D peaks at 250 nA, indicating a strong component of proton conductivity (Fig. 2B, red trace). This proton conductivity is consistent with the activation energy for the conductivity of shark AoL jelly (11). With PdH_x contacts, I_D drops over time. At first sight, this time dependence may suggest proton-blocking behavior at the contact. However, we observed similar time dependence when measuring highly proton-conducting materials such as Nafion with PdH_x contacts (14). This time dependence arises when the diffusion of hydrogen to the PdH_x contact surface is slower than the conduction of H⁺ in the proton-conducting material (14).

We also measured the proton conductivity (σ_H+) of AoL jelly extracted from big skate (R. binoculata) and bonnethead shark (S. tiburo) (fig. S3). The AoL jelly for all species is proton-conducting with a fivefold range in σ_H+. The AoL jelly from big skate is more conducting than the AoL jelly from bonnethead shark, which is, in turn, more conducting than the AoL jelly from longnose skate. The time dependence of I_D is consistent with the differences in σ_H+ and our previous observations with Nafion (14). Although it is likely that some differences in σ_H+ across species are intrinsic to the AoL jelly, the small differences in σ_H+ measured with these devices may also arise because of variance in device geometry. It would be certainly of interest to perform a systematic study of the AoL jelly conductivity across species, as well as age and gender of the subjects.

The injection of protons from the contact into the AoL jelly incurs a significant contact resistance, and the depletion of H at the PdH_x contacts causes a drop in I_D as observed in our previous work (14). To circumvent these issues, we used a modified four-point probe geometry to measure the proton conductivity (σ_H+) of the AoL jelly independently of the PdH_x–AoL jelly contact resistance. In this geometry, we added two thin Au contacts between the Pd source and drain, for a total of four contacts. When we apply V_SD to the PdH_x source and drain, a current of H⁺ flows in the AoL jelly, as demonstrated in the standard two-probe geometry. We measured the potential difference between the two Au contacts. This potential difference is proportional to I_D and inversely proportional to the conductivity of the AoL jelly (Fig. 2C). Because no current flows across the Au–AoL jelly interface, there is no potential drop and the PdH_x contact resistance does not affect the measurement of the conductivity of the AoL jelly. For Pd contacts at 90% RH, we find σ_ion ≈ 100 to 1000 nS/cm depending on V_SD (Fig. 2D, blue trace), which is consistent with our observations in the two-probe geometry (Fig. 2B). For PdH_x contacts at 90% RH, we find σ_H+ ≈ 6 to 200 μS/cm, peaking at 200 μS/cm for V_SD = 1 V (Fig. 2D, red trace). This conductivity is similar to that of dialyzed AoL jelly from a shark in a dc measurement (800 μS/cm) (11). In our measurements, the proton conductivity of the AoL jelly depends exponentially on V_SD, which is similar to the highly proton-conducting polymer Nafion (19). To confirm that the AoL jelly conductivity arises from H⁺, we repeated the measurements by hydrating the sample with deuterated water and by exposing the Pd to deuterium rather than to hydrogen. Deuterium ions (D⁺) transport along hydrated materials in a similar fashion as protons, but with lower mobility and associated lower conductivity (20). Hydrating the AoL jelly samples with D₂O substitutes D⁺ for H⁺ in the material. A subsequent decrease in conductivity for D⁺ (σ_D+)—the kinetic isotope effect—is a well-accepted signature for conductivity arising predominantly from protons (20). For the AoL jelly, σ_D+ = ¹/₂σ_H+ for any V_SD when measured in the four-point probe geometry (Fig. 2D, green trace). The kinetic isotope effect with this four-point probe measurement unequivocally points to proton conduction in the AoL jelly (20). However, these measurements likely underestimate the AoL jelly conductivity (σ_H+ = 0.2 mS/cm). In the four-point probe geometry, we find the proton conductivity of Nafion at σ_H+ = 2 mS/cm, which is lower than the literature value (σ_H+ = 78 mS/cm) (21). This measured conductivity of the Nafion control sample is smaller than expected. We suspect that non-ideal geometry is the cause of this variation. The calculated conductivity assumes that only the material in the channel is relevant to conduction, because the electric field is highest here. However, if the interface resistance is comparable to the channel resistance, this assumption may not be accurate, skewing the calculated results (22). To address this variation, we measured the proton conductivity of the AoL jelly in a transmission line geometry.

We constructed devices with different lengths between the Pd source and the drain to estimate the proton conductivity of the AoL jelly (Fig. 2E). This varying source-drain length (L_SD) geometry is commonly known as a transmission line measurement (TLM) (Fig. 2E) (23). We applied V_SD = 1 V on devices with L_SD ranging from 0.25 to 5.0 mm, measured I_D, and calculated the resistance of each device, R_L. Because different devices contained AoL jelly of different thicknesses, we multiplied R_L by the AoL jelly thickness to get the normalized resistance R_LN and plotted R_LN as a function of L_SD (Fig. 2F). In this geometry, the resistance of the AoL jelly increases linearly with L_SD, but the contact resistance at the source–AoL jelly and drain–AoL jelly interface is constant. To extrapolate the resistivity of the AoL jelly, we plot R_LN as a function of L_SD. The slope of this plot is proportional to the resistivity of the AoL jelly, and the intercept on the R_LN axis for L_SD = 0 is the contact resistance. For the AoL jelly, we obtain σ_H+ = 1.8 ± 0.9 mS/cm, which is only one order of magnitude lower than the proton conductivity of Nafion σ_H+ = 28 ± 14 mS/cm measured in the same geometry (fig. S4). The proton conductivity of the Nafion control sample (28 ± 14 mS/cm) measured in a TLM geometry is much closer to the reported value of 78 mS/cm (21). Therefore, we conclude that σ_H+ = 1.8 ± 0.9 mS/cm measured for the AoL jelly in the TLM geometry is our best estimate of the proton conductivity of the AoL jelly.

When working with biological materials, characterization is often challenging because many components are present and further processing to separate these components may change the materials properties and function. Here, we chose to perform a preliminary analysis on the as-extracted AoL jelly to look for the components that might provide the high protonic conductivity. Although the AoL jelly likely contains a mixture of proteins and polyglycans, we focus on the sulfated polyglycan keratan sulfate (KS) (Fig. 3A) following an early report by Doyle (24). We characterized the AoL jelly from the skate (R. binoculata) using monosaccharide compositional analysis and Fourier transform infrared (FTIR) spectroscopy. In the monosaccharide composition analysis, we find a nearly 1:1 ratio of glucosamine to galactose (Fig. 3B). This ratio is consistent with the presence of KS in the AoL jelly and corroborates Doyle’s analyses (24). However, the presence of other sulfated polyglycans or polysaccharides cannot be completely ruled out at this time. The FTIR spectrum of the AoL skate jelly also contains the signature peaks of KS (Fig. 3C), including the peak at 1238 cm⁻¹ attributed to S=O vibrations of the sulfate group (25). The sulfate group in the KS is an acid [pK_a (where K_a is the acid dissociation constant) = 2] (26), which donates the protons that contribute to the proton conductivity of the material (13, 15). By comparison, the sulfonic group in Nafion is a stronger acid, so Nafion has correspondingly higher charge carrier density and proton conductivity (21). However, proton conduction requires both a high carrier density and a high carrier mobility, so creating pathways for proton transport is also essential. KS has several hydrophilic groups that may induce the organization of water in the AoL jelly into hydrogen bond chains, allowing proton conduction according to the Grotthuss mechanism (27). Chitin-derived polysaccharides functionalized with acid and base groups are also good proton conductors and have chemical similarities to KS (12, 13). Charged amino acids in the squid protein reflectin also contribute to its high proton conductivity (28, 29). Further investigations into the composition and microstructure of the AoL jelly and its correlation to the AoL jelly proton conductivity across species are necessary to create a complete picture of the AoL high proton conductivity.

We extracted AoL jelly from freshly caught and recently expired R. binoculata (big skate), R. rhina (longnose skate), and S. tiburo (bonnethead shark). AoL jelly was pressed from visible surface pores and collected with a mechanical plunger-style pipette. Samples were stored at −20°C until use. Just before measurement, the AoL jelly was thawed at room temperature and then spun in a centrifuge at 6000 rpm to remove air bubbles. The AoL jelly was then drop-cast onto substrates using a syringe. Because of the thick, viscous nature of the AoL jelly, precise volume control was difficult.

Electrical measurements were performed on a Signatone H-100 probe station using a custom atmospheric isolation chamber. Dakota Instruments mass flow controllers were used to set the ratio of dry nitrogen, wet nitrogen, hydrogen, and deuterium controlled by a LabView DAQ. An Agilent 4155C semiconductor parameter analyzer was used for all electrical measurements.

Two-terminal measurements were performed on Si substrates with a 100-nm SiO₂ layer. Pd contacts were deposited by conventional photolithography and e-beam evaporation. Contacts were 30 μm wide with a 1-μm separation. The Pd contacts were 60 nm thick with a 5-nm Cr adhesion layer. The AoL jelly layer was typically 50 μm thick.

Samples were saturated with either water (H₂O) and hydrogen gas (H₂) or deuterated water (D₂O) and deuterium gas (D₂). Samples were held for 30 min after switching to allow the diffusive exchange of ions.

Samples consisted of 100-nm Pd contacts with a 5-nm Cr adhesion layer deposited on glass slides. Mylar shadow masks were used to define 5-mm-wide contacts with L_SD = 0.25, 0.5, 0.75, 1.0, 2.0, 3.0, 4.0, and 5.0 mm. The sample thickness was usually around 1 mm. Measurements were performed at V_SD = 1 V.

Samples were fabricated on Si wafers with a 100-nm SiO₂ layer. Conventional photolithography was used to separately pattern Pd and Au contacts. Pd contacts were 30 μm wide and 60 nm thick and were separated by a 6-μm gap. Two Au contacts were deposited between the Pd contacts. Au contacts were 50 nm thick and 1 μm wide and were separated by a 1.5-μm gap. Both Pd and Au contacts had a 5-nm Cr adhesion layer. The AoL jelly layer was typically 50 μm thick.

An AoL jelly sample was taken from a freshly caught and recently expired skate (R. binoculata). This sample was separated into 0.5-ml aliquots and frozen at −80°C. One of the aliquots was sent to the Biotechnology Core Resources Laboratory in San Diego and used for monosaccharide compositional analysis via high-performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD). The sample (0.1 mg) was treated with 200 μl of 2 M trifluoroacetic acid at 100°C for 4 hours to cleave all glycosidic linkages and processed further for monosaccharide composition and detection. This analysis method breaks down polymers into monomers and, in the process, also breaks down any sulfate and acetyl side chains. Monosaccharide standards were treated in a parallel fashion and used for calibration of HPAEC-PAD.

FTIR spectra were recorded with a Bruker VERTEX 70 FTIR spectrophotometer (4000 to 400 cm⁻¹, 4 cm⁻¹ resolution) working in attenuated total reflectance (ATR) mode. One droplet of skate jelly was carefully placed on the ATR crystal and allowed to dry for 30 min. Then, we recorded the FTIR spectrum.