# Mechanical Properties of DNA Hydrogels: Towards Highly Programmable Biomaterials
## 5 Dissecting the Mechanical Properties of DNA Hydrogels  
### Characterization Methods  
There are a variety of methods to characterize the mechanical properties of hydrogels; generally categorized as compression, indentation, and rheology. Conventional rheological measurements are conducted via bulk rheology (Figure 4a), which includes parallel plate [33; 54; 64] and parallel cone and plate [51]. Given the innate thermo-reversibility of DNA hydrogels, temperature-controlled rheology is often of interest. Bulk rheology is advantageous because it is a well-established technique; however, it is limited to low frequencies (<100 Hz), often requires a large sample volume, and cannot probe the heterogeneity of the sample. The required large sample volume, typically in the milliliter range, is an important factor for the use of bulk rheology to characterize pure DNA hydrogels due to current material cost. In contrast to bulk rheology, microrheology utilizes a much smaller sample volume, typically less than 100 \(\upmu\)L. Microrheology uses the motion of embedded probe particles caused by either thermal  
Figure 4: Common characterization methods for DNA hydrogel. (**a**) Bulk rheology and dynamic moduli. (**b**) Graphical depiction of microrheology showing constrained probe particle diffusion within the hydrogel network. The probe mean squared displacement (MSD) is used to determine the material behavior. (**c**) Network vibrations and interparticle hybridization rupture measured by dynamic light scattering (DLS) where a fast and slow decay time are observed and correspond to the timescale for each event, respectively. (**d**) Probe-based force controlled nanoindentation of DNA-based hydrogel to determine the material behaviors. fluctuation (passive) or externally applied forces (active) to determine the rheological properties of the sample (Figure 4b). Compared to bulk rheology, microrheology offers access to a broader frequency range (10\({}^{-1}\)-10\({}^{6}\) Hz) and the potential to study sample heterogeneity [101; 102; 103]. The measurement techniques for passive microrheology include dynamic light scattering (DLS), diffusive wave spectroscopy (DWS) and particle tracking microscopy (PTM) [62; 101; 104; 105]. Since the relaxation time for a hydrogel might be longer than the timeframe allowed by DLS or DWS (\(\sim\)10 s) PTM or bulk rheology can be used in conjunction to access the lower frequency regime (<0.1 Hz) [106]. DLS measurements are also dependent on sufficient scattering from the probe particles to mask the scattering signal from the sample, though they typically require low probe concentrations. For samples of greater opacity DWS is a viable alternative, although it requires a much higher density of probe particles which may interfere with the properties of the hydrogel. However, DWS is also more sensitive at higher frequencies compared to DLS [101; 103; 104]. Alternatively, DLS can be used without probe particles to study the network vibrations and reorganization at multiple length-scales (Figure 4c) [107]. Among the other techniques available to characterize soft materials, nanoindentation represents one of the most convenient methods for the direct measurements of mechanical properties of hydrogels at different scales [108]. This technique relies on the application of a controlled force (load) on the surface of a soft biomaterials with a nanoprobe to induce local deformation (Figure 4d). The depth of indentation or deformation as well as the response time for a given load are monitored and used to identify mechanical properties of the sample. Nanoindenter instrument and atomic force microscope (AFM) are the two types of instruments that can be used to perform nanoindentation on these soft biomaterials [108; 109]. In addition, AFM enables the combination of nanoscale imaging and mechanical characterization of biomaterials that are used to scaffold cells for biomedical applications such as tissue engineering.