Hydrogel

A hydrogel matrix is the cornerstone of SporeScope’s functionality, enabling three-dimensional spatial immobilisation of bacterial spores while maintaining optical transparency for real-time observation. The hydrogel acts as a biocompatible scaffold, simulating the native environment spores would encounter in vivo.

Importantly, SporeScope is compatible with a wide range of hydrogels, allowing researchers to tailor the internal microenvironment to match specific biological contexts — whether mimicking soil, gut, or medical implant conditions. This flexibility makes the platform a powerful tool for studying bacterial behaviour across disciplines including agriculture, biomedicine, and environmental microbiology.

Why We Chose Alginate

In our early experiments, we selected alginate hydrogel due to its excellent compatibility with biological samples and ease of gelation:

  • Mild Crosslinking Conditions: Alginate crosslinks in the presence of calcium ions (Ca²⁺) at room temperature, avoiding the need for toxic or harsh chemicals. This makes it safe for use with dormant bacterial spores, ensuring experimental reliability from the start.

  • Customizable Porosity: By adjusting the alginate concentration, we control pore size—allowing nutrient diffusion while maintaining spore immobilization. This tunable structure supports both germination and high-resolution imaging.

  • Enhanced Stability: Unlike short-lived hydrogels, alginate forms a mechanically stable matrix that lasts throughout long-term experiments. This stability is crucial for continuous, time-lapse fluorescence microscopy.

  • Immobilization for Accuracy: One of the primary reasons we used a hydrogel at all was to keep bacterial spores fixed in place. In liquid environments, spores drift and signals overlap, making it difficult to monitor single-cell behavior. Alginate eliminates this issue by creating a scaffold that holds each spore in place while allowing nutrients and stimuli to diffuse freely.

Transitioning to PEGDA for the Final Prototype

As our design evolved, we identified poly(ethylene glycol) diacrylate (PEGDA) as the optimal hydrogel for full chip integration. PEGDA can be UV-crosslinked, allowing us to precisely localize and pattern hydrogel formation directly within our microfluidic device:

  • Photocrosslinking for On-Chip Precision: PEGDA gels can be polymerized in situ using ultraviolet light. This gives us unparalleled spatial control during chip fabrication and removes the need to introduce chemical crosslinkers post-fabrication.

  • Biocompatibility and Optical Clarity: Like alginate, PEGDA is biocompatible. It also offers high transparency, ideal for real-time confocal fluorescence microscopy. This ensures that spores can be visualized clearly at single-cell resolution.

  • Improved Mechanical Strength: PEGDA creates a more robust network that withstands fluid flow and long-term imaging better than alginate, making it more suitable for reusable, integrated systems.

By starting with alginate to validate our concept and transitioning to PEGDA for full-chip realization, we ensured that our tool combines biological relevance with engineering precision—enabling controlled, real-time monitoring of bacterial spore germination within a dynamic, tunable microenvironment.