Characterising DNA film structure

We first characterise the structure of films formed by drying aqueous DNA solutions on a glass coverslip. At macroscopic length scales, all the films appear homogeneous, however scanning electron microscopy (SEM) reveals that the films exhibit topographical heterogeneities at sub-micron length scales. Figure 1(a) and (b) show scanning electron micrographs of a 0.0031 mg/mm2 surface density DNA film that has only been exposed to ambient light. The film is comprised of closely packed, discrete and multi-faceted crystals. Figure 1(c) and (d) show SEM images of a 0.0031 mg/mm2 film after exposing it to a dosage of 320 J/cm2 UVA light (peak narrowband emission at 365 nm). Figure 1(e) and (f) show complementary images of a 0.0031 mg/mm2 film after exposure to an equivalent dosage of UVB light (peak narrowband emission at 302 nm). Table 1 details the average crystal size for UVA and UVB irradiated 0.0031 mg/mm2 surface density films along with 0.0031 mg/mm2 and 0.0124 mg/mm2 surface density control films exposed only to ambient light. Both UVA and UVB exposure induce statistically significant reductions in crystal size relative to films exposed only to ambient light, however the greatest reduction occurs with UVA irradiation. Increasing the film density also increases the average crystal size.

Figure 1 DNA film structure. Scanning electron micrographs of (a,b) a 0.0031 mg/mm2 surface density DNA film exposed only to ambient UV light, (c,d) a 0.0031 mg/mm2 DNA film irradiated with a dosage of 320 J/cm2 UVA light (365 nm), and (e,f) a 0.0031 mg/mm2 DNA film irradiated with a dosage of 320 J/cm2 UVB light (302 nm). Scale bars in the left and right columns of images respectively denote 2 μm and 400 nm. Full size image

Table 1 DNA Film Crystal size variation with surface density and UV irradiation. Full size table

Characterising the photonic properties of DNA films

We next interrogate the photonic properties of DNA films coated on UV transparent coverslips by measuring their absorbance and transmission spectra. Figure 2(a) shows that the films do not absorb light at wavelengths greater than 500 nm, therefore measured spectra are truncated to show only UV and visible blue wavelengths. All of the films absorb UV light strongly in the UVC range (200–280 nm) and exhibit an abrupt reduction in absorbance over the UVB range (280–315 nm). Increases in film surface density delay the onset of this transition to longer UVB wavelengths. Within the UVA range (315–400 nm), 0.0031 and 0.0124 mg/mm2 surface density films absorb little light, however 0.031 mg/mm2 films show notable UVA absorbance. Figure 2(b) highlights that all films reduce UVC transmission by greater than 90% at certain wavelengths. Increases in film surface density result in decreased transmission in the UVA, UVB, and UVC ranges. The most notable changes occur within the range \({\rm{300}}\le \lambda \le {\rm{450}}\) nm.

Figure 2 Surface density effects on UV absorbance and transmission. Average (a) absorbance and (b) transmission spectra of DNA films with surface densities of 0.0031 mg/mm2 (n = 3, open circle), 0.0124 mg/mm2 (n = 3, open diamond) and 0.031 mg/mm2 (n = 3, open square) plotted against wavelength in the range \({\rm{200}}\le \lambda \le {\rm{450}}\) nm. Open triangle symbols denote the average (n = 3) absorbance and transmission spectra of the optically transparent glass coverslips on which all DNA films are coated. Vertical lines delineate the UVA (315–400 nm), UVB (280–315 nm), and UVC (200–280 nm) ranges. Standard deviations in all cases are smaller than the symbol size and have not been plotted to improve visual clarity. Full size image

Characterising changes in DNA film photonic properties with UV irradiation

Changes in the photonic properties of DNA films after UV irradiation are subsequently assessed. The absorbance and transmission spectra of films only exposed to ambient light are first captured. The films are then sequentially irradiated with increasing dosages of either UVA or UVB light using a UV lamp. After each dosage, the film spectra are re-quantified. The UV lamp increases the local air temperature around the films to no greater than 27 °C, substantially below the temperature required to denature DNA43 (>80 °C). Figure 3 shows changes in the average absorbance and transmission spectra of DNA films after exposure to UVB dosages of 80, 160 and 320 J/cm2 (n = 3 individual films for each dosage). A UVB dosage of 160 J/cm2 has previously been reported to damage the barrier function of human stratum corneum 5 skin tissue. As shown in Fig. 3(a) and (b), increases in UVB dosage cause 0.0031 mg/mm2 films to increase transmission and decrease absorbance within the UVC range. A small decrease in UVA transmission is also observed. In contrast, films with surface densities of 0.0124 and 0.031 mg/mm2, shown in Fig. 3(c) through (f), show little variation in UV transmission at wavelengths less than 300 nm, but notable decreases at longer wavelengths. Reductions in UV transmission scale monotonically with UVB dosage except 0.031 mg/mm2 films irradiated with a 80 J/cm2 dosage. Relative to controls, this dosage causes a small decrease in transmission around 300 nm. Decreases in UVA transmission with UVB dosage become more pronounced as film surface densities increase.

Figure 3 Effects of UVB irradiation. Absorbance (left column) and transmission (right column) plotted against wavelength, λ, for films with a surface density of 0.0031 mg/mm2 (top row), 0.0124 mg/mm2 (middle row) and 0.031 mg/mm2 (bottom row). Each of the panels (a–f) display the average (n = 3 individual films for each dosage) absorbance or transmission spectrum prior to UVB irradiation (Control: red circle) and after UVB dosages of 80 J/cm2 (green triangle), 160 J/cm2 (black inverted triangle) and 320 J/cm2 (blue square). Standard deviations in all cases are smaller than the symbol size and have not been plotted to improve visual clarity. Full size image

Figure 4 shows variations in the absorbance and transmission spectra of films with different surface densities after irradiation with UVA dosages of 80, 160 and 320 J/cm2. Increases in UVA dosage for the 0.0031 mg/mm2 films result in notable decreases in UV transmission at wavelengths greater than 300 nm and small decreases in absorbance at wavelengths below 300 nm. In contrast with the UVB irradiation results in Fig. 3, reductions in UVA and UVB transmission with UVA dosage become less pronounced with increases in film surface density.

Figure 4 Effects of UVA irradiation. Absorbance (left column) and transmission (right column) plotted against wavelength, λ, for films with a surface density of 0.0031 mg/mm2 (top row), 0.0124 mg/mm2 (middle row) and 0.031 mg/mm2 (bottom row). Each of the panels (a–f) display the average (n = 3 individual films for each dosage) absorbance or transmission spectrum prior to UVA irradiation (Control: red circle) and after UVA dosages of 80 J/cm2 (green triangle), 160 J/cm2 (black inverted triangle) and 320 J/cm2 (blue square). Standard deviations in all cases are smaller than the symbol size and have not been plotted to improve visual clarity. Full size image

In order to characterise overall changes in UV transmission through the films after UVA or UVB irradiation, the fraction of total incident light transmitted, or transmittance44, \(\tau ={\int }_{{\lambda }_{{\rm{\min }}}}^{{\lambda }_{{\rm{\max }}}}\tau (\lambda ){{\rm{\Phi }}}_{\lambda i}d\lambda /{\int }_{{\lambda }_{{\rm{\min }}}}^{{\lambda }_{{\rm{\max }}}}{{\rm{\Phi }}}_{\lambda i}d\lambda \), is quantified over the UVA (\({\lambda }_{\max }=400\) nm, \({\lambda }_{\min }=315\) nm), UVB (\({\lambda }_{\max }=315\) nm, \({\lambda }_{\min }=280\) nm) and UVC (\({\lambda }_{\max }=280\) nm, \({\lambda }_{\min }=200\) nm) ranges. Here, \({{\rm{\Phi }}}_{\lambda i}\) denotes the incident spectral flux and \(\tau (\lambda )\) denotes the spectral transmittance; the ratio of the transmitted spectral flux to the incident spectral flux at each wavelength. Figure 5(a–c) respectively show how the transmittance of light in the UVA, UVB and UVC ranges differ with film surface density and UVB dosage. Increases in UVB dosage cause notable reductions in transmittance primarily within the UVA range. Relative to controls exposed only to ambient light, the 0.031 mg/mm2 films exhibit the largest decrease from τ = 0.76 ± 0.05 to τ = 0.64 ± 0.02 and τ = 0.54 ± 0.02 after UVB dosages of 160 and 320 J/cm2 respectively. The uncertainties here correspond to standard deviations. Smaller changes in UVA transmittance are observed for films with lower surface densities. Dosages of 320 J/cm2 cause transmittance reductions of Δτ = −0.1 in 0.0031 mg/mm2 films and Δτ = −0.13 in 0.0124 mg/mm2 films. UVB dosages of 160 J/cm2 or greater also marginally reduce UVB transmission for 0.0124 and 0.031 mg/mm2 films. Small increases in UVC transmittance are observed only for the 0.0031 mg/mm2 films.

Figure 5 Effect of UVB dosage on UV film transmittance. Average DNA film transmittance, τ, in the (a) UVA range (315–400 nm), (b) UVB range (280–315 nm) and (c) UVC range (200–280 nm) prior to UVB exposure (open bar) and after UVB irradiation with dosages of 80, 160 and 320 J/cm2 (respectively denoted by increasingly dark bar colors). Error bars denote standard deviations of n = 3 individual films. Full size image

Figure 6(a), (b) and (c) respectively show how the transmittance of light in the UVA, UVB and UVC ranges differ with UVA dosage. With a dosage of 320 J/cm2, fractional UVA film transmittance decreases by Δτ = −0.18, −0.16, and −0.16 for the 0.0031, 0.0124, and 0.031 mg/mm2 surface density films respectively. Notable decreases in UVB film transmittance also occur, but only for the 0.0031 mg/mm2 film, where a dosage of 320 J/cm2 reduces UVB transmittance by Δτ = −0.24. UVA irradiation also marginally increases UVC transmission for films with surface densities of 0.0124 mg/mm2 or greater. Dosages of 80 J/cm2 increase UVC transmittance by \(0.06\le {\rm{\Delta }}\tau \le 0.1\), however subsequent irradiation does not alter transmittance further.

Figure 6 Effect of UVA dosage on UV film transmittance. Average DNA film transmittance, τ, in the (a) UVA range (315–400 nm), (b) UVB range (280–315 nm) and (c) UVC range (200–280 nm) prior to UVA exposure (open bar) and after UVA irradiation with dosages of 80, 160 and 320 J/cm2 (respectively denoted by increasingly dark bar colors). Error bars denote standard deviations of n = 3 individual films. Full size image

Characterising DNA film swelling and evaporation kinetics

Studies exploring the evaporation kinetics of DNA films when topically applied to human skin are additionally performed to further probe their structure and hygroscopic properties. Isolated samples of identically sized human stratum corneum (SC, surface area A = 1440 mm2), the outermost layer of skin that governs trans epidermal water loss45 are adhered to an elastomer coated aluminium substrate. Samples are then uniformly coated with 500 μl of an aqueous DNA solution, then desiccated for 24 hr at 10% R.H to establish the total mass of SC sample, DNA film and substrate, M 0 . Once fully rehydrated, changes in the sample mass, M(t), are recorded over a 3.5 hr period within a controlled 24% relative humidity (R.H.) environment. This period is sufficiently long to observe drying behavior46, 47. Figure 7(a) displays changes in the area scaled average water mass of the SC and DNA films over time, \({M}_{W}(t)=(M(t)-{M}_{0})/A\). The fully hydrated area scaled water mass, M W (0), increases with film surface density. Subsequent drying over the first 60 min causes all coated and uncoated samples to rapidly dehydrate. Thereafter, the rate of drying decreases. After the first 20 min of drying, samples with 0.00173 mg/mm2 coatings show no difference in M W relative to control samples coated only with deionized water (DIW). Higher surface density films however result in a larger M W throughout the 3.5 hr drying period. In order to quantify the effects of the ethanol used to create the DNA solutions, drying behavior of SC samples coated with 6% aqueous ethanol solutions are also assessed. These samples exhibit a reduced water mass relative to DIW coated controls over the first 2 hr of drying. Thereafter, they become comparable. This indicates that the ethanol reduces rather than increases the ability of SC to hold water in a hydrated state.

Figure 7 Evaporation kinetics of DNA film coated SC. (a) Average area scaled water mass, M W , of control and DNA film coated SC samples drying over time in a 24% R.H. environment. Control results include SC samples coated with DIW water (black triangle) and 6% aqueous ethanol solutions (white triangle). DNA film coating results include surface densities of 0.00173 (red circle), 0.00347 (yellow inverted triangle), 0.00694 (green diamond), and 0.0174 mg/mm2 (blue square). Shaded regions denote standard deviations of n = 3 individual SC samples. (Inset) Average mass swelling ratio, Q m , plotted against DNA film surface density, \(\Gamma \). Error bars denote standard deviations of n = 3 individual SC samples. When not visible, errors are smaller than the symbol size. (b) Results from panel (a) scaled by the DIW coated control results (black diamond). Shaded regions denote propagated standard deviations. Full size image

The inset figure in Fig. 7(a) plots changes in the DNA film mass swelling ratio, Q M , against film surface density, Γ. This ratio denotes the quotient of the maximum swollen DNA film mass to the dry mass of the DNA film alone48. The swollen mass of the DNA films are obtained by subtracting the average fully hydrated mass of uncoated SC samples from the average hydrated mass of SC samples coated with the DNA films. This swelling ratio is commonly used to characterise hydrogel behavior, where swelling of the hydrogel is eventually balanced by the elastic restoring forces of the crosslinked polymer network48. The data indicates that as Γ increases, Q M decreases and the maximum area scaled water mass that the film can absorb is approached. To verify this trend, the swelling ratio of 0.0348 mg/mm2 films are additionally measured and included in the results.

To further highlight the impact of the DNA films on the rate of cutaneous water loss, Fig. 7(b) shows the results of Fig. 7(a) scaled by the average area scaled water mass of DIW coated controls. Over the first 60 min of drying, samples coated with a DNA film exhibit increased rates of evaporation relative to controls. This rapid evaporation quickly eliminates the elevated water masses within the fully hydrated film coated SC samples. The rate of this evaporation also increases with film surface density. Thereafter, the evaporation rate of samples coated with a 0.0017 mg/mm2 film remains near consistent with control samples. However, the three highest surface density films exhibit water evaporation rates smaller than SC samples without a film coating. This diminished rate of evaporation also scales monotonically with the film surface density. These results are consistent with previous studies of drying behavior in polymeric films49. For intermediate and high concentrations of a polymer in a solvent, the diffusion coefficient of the solvent across the drying film, which is directly related to the evaporation rate, decreases as the polymer concentration is increased. In contrast, as the polymer concentration is decreased towards very dilute systems, the diffusion coefficient plateaus and becomes consistent with that of the solvent alone. Relative to controls, the time varying change in water evaporation rate of the three highest surface density films after 60 min drying also suggests that as water evaporates, the DNA mass fraction within the films increase, further slowing drying.