Poly(ethylene glycol)-1000-dimethacrylate (PEG-1000- DMA) was from Polysciences (Warrington, PA, USA). 2- Hydroxy-2-methyl-1-phenyl-propan-1-one (Darocur^® 1173) was from Ciba Specialty Chemicals (Basel, Switzerland). Phosphate buffered saline (PBS) was from Sigma Aldrich Denmark (Brøndby, Denmark).

The hydrogel was prepared as in [10,16]. Briefly, the hydrogel precursor solution (HPS) consisted of 65 mM PEG-1000-DMA and 10 mM Darocur^® 1173 (a photoinitiator) in PBS. We will refer to this solution as 1000P. Free-radical polymerisation of 1000P was initiated by 4, 6, 8, 10 or 15 min of photocrosslinking of the HPS by UV light from an EA-140 UV lamp with wavelength λ = 365 nm (Spectroline, Westbury, NY, USA) at a lamp distance of 1 cm. The lamp intensity at a distance of 15 cm was 800 µW·cm^–2. We will refer to the crosslinked gel as X1000P.

Equilibrium swelling experiments were used to determine the structural characteristics of the PEG-DMA gels. Irradiated hydrogel samples were prepared from 3 ml of HPS per sample. Excess liquid was poured off and the gels were allowed to equilibrate at ambient temperature (~25˚C) for 24 h. Thereafter, the gels were weighed to determine the irradiated gel weight, W_ir. Then they were dried under vacuum for 24 h to remove all water (the dried gel mass corresponded to the expected dry mass of PEG-1000-DMA and Darocur in 3 ml of HPS) and weighed to determine the dry gel weight W_d, as in [17]. The percentage of water content in the irradiated gels was calculated as (W_i – W_d)/W_ir·100% [18]. The dried gels were rehydrated in PBS at ambient temperature (~25˚C) for 24 h. Excess liquid was poured off and the gels were allowed to equilibrate at ambient temperature (~25˚C) for 24 h. Then the gels were weighed to determine the swollen gel weight, W_s and percentage of water content determined as (W_s – W_d)/W_s·100%.

Fourier transform infrared (FTIR) spectroscopy was performed on a Thermo Nicolet Nexus 870 with a Ge 45˚ Smart Ark attenuated total reflectance (ATR) crystal from Thermo Scientific, MA, USA. The FTIR spectra were recorded using OMNIC software from Thermo Nicolet Corporation with an average of 32 scans in the range 650 - 4000 cm^–1 and at a resolution of 4 cm^–1. Samples were dried in a vacuum dessicator for 24 h prior to analysis in order to reduce the contribution from water in the recorded spectrum.

Raman spectra were obtained as in [19] by use of a DILOR-XY 800 mm focal length multichannel spectrometer with horizontal Nd:YVO₄ laser excitation (532.8 nm, ~300 mW, vertically polarized). Rayleigh-scattered light was filtered off with a double pre-monochromator (slit width 500 µm). The Raman light was collected at 90˚ scattering, dispersed by use of an 1800 lines/mm grating, and focused onto a charge-coupled device (CCD) detector, cooled to 140 K by liquid nitrogen. The spectral resolution was approximately 4 cm^–1. Hydrogel samples were prepared as described above and were not dried prior to measurements. The recorded spectra were normalized to obtain the same intensity for the spectral peak at 1470 cm^–1 (attributed to H-C-H bending). The relative number of C=C bonds in the gels was obtained by comparing the peak height at 1640 cm^–1 (attributed to C=C stretching), which was used as a measure of the degree of crosslinking.

The test cell for impedance spectroscopy measurements consisted of a Teflon support with two Pt electrodes between which a cylindrical crown rubber support (approximately 2.2 mm height and 1.5 cm diameter) filled with gel sample was placed and clamped as previously described [20]. The electrodes were connected to a Frequency Response Analyzer (Solartron 1260, Farnborough, England) and measurements were recorded for 100 data points in the frequency range of 1 Hz - 10 MHz, at a maximum voltage of 0.01 V.

The water content of the gel influences the mechanical integrity and strength of the gel as well as the diffusion of nutrients and metabolites through the gel to a cell/ biomimetic membrane [7]. Controlling the gel water content is important for the gel’s interaction with hydrogelencapsulated biomimetic membranes, such as those presented in our earlier work [10]. The high water absorbance of hydrogels is one of their most important material properties and therefore, it is crucial to be able to tailor the water content of the gels to suit the application that they are intended for.

We found that the irradiated and equilibrated PEGDMA hydrogels contained ~92 wt% water independent of UV irradiation time, see Figure 2. However, after the gels were dried and rehydrated overnight, the water uptake ability of the gels increased with an increase in the photocrosslinking time with a water content ranging from 50% for samples irradiated for 4 min to 90 wt% for samples irradiated for 15 min approaching the water content of the non-dried gels, see Figure 2.

An explanation for the increased water uptake ability with increased UV irradiation could be that drying the gels causes partial reversible collapse of pores in the gel meshwork as has been observed for similar gels by Wu et al. [17], with an increased degree of pore collapse for shorter crosslinking times. Increasing the crosslinking time would increase the crosslinking density and thereby improve the stability of the gel meshwork, leading to less pore collapse. Less pore collapse entails more pores that can be filled with water and thus more water absorption.

Interestingly, Andreopoulos et al. [21] report that the water content in a PEG-based gel decreases with increased photocrosslinking time. It is thus possible to tailor the water content of PEG-DMA hydrogels by selecting a suitable UV photocrosslinking time.

In order to analyze whether this trend corresponded to an increase in crosslinking with increased UV irradiation time, we studied samples with FTIR and RS. With FTIR we compared spectral peaks at 1680 - 1780 cm^–1 (assigned to C=O bond vibrations and indicated by the label “a” in Figure 1) and with RS spectral peaks at 1640 cm^–1 (assigned to C=C bonds and indicated by the label “b” in Figure 1) [22]. These bands originate from the methacry-

late groups, which form covalent bonds through an addition reaction during the polymerization. In the addition reaction, the alkene functionality is converted to an alkane functionality. Therefore, the number of C=C bonds can be used as a measure of the degree of crosslinking of the polymer meshwork. In FTIR, the C=C peak overlapped a peak from water, which restricted quantification of the relative number of C=C bonds to dry gels. Therefore we used Raman spectroscopy (RS) to quantify C=C bonds in the wet gel, due to the low Raman scatter from bulk water.

A physical parameter of great interest in determining sample changes is the specific electrical resistance, which can be determined from impedance spectroscopy (IS) measurements using equivalent circuits as models [26, 27]. We collected the impedance plots for the hydrogel at different photocrosslinking times, and clear differences could be observed as shown in Figure 5(a). The fitting of the impedance data by using a non-linear program [28] allows determination of the electrical resistance of the different gel samples (R_G), which can be converted to specific electrical resistance, ρ, using ρ = R_G·A/l, where A = 177 mm² is the surface area of the gel sample and l = 2.2 mm is the thickness of the gel. Specific electrical resistance values obtained for gel samples are shown in Figure 5(b). The electrical resistance of the gels ranged from 0.7 Ωm for 4 min photocrosslinking to 3.5 Ωm for 15 min photocrosslinking. For comparison, PBS has a resistivity of 8.4 Ωm (Bäckström & Hélix-Nielsen, unpublished results) and deionized water a resistivity of 1.8·10⁵ Ωm [29].

This showed that the gels had a low resistivity (and a high conductivity), making them suitable for use in ion channel-containing hydro gel-encapsulated membranes for biosensor applications. An increase in the specific electrical resistance as a result of an increase in the photocrosslinking time was obtained, which correlated with an increase in the crosslinking density of the gel network. It is remarkable that an increase in photocrosslinking increased the water absorption of the gel, while also increasing its resistivity. We attribute the increased gel resistance to the presence of the hydrophobic initiator Darocur consistent with previous work [16].