General Overview - Engineering Routine from Laboratory Biosensor to On-site Test Kit

      In order to make engineered biosensor cells suitable for on-site testing, we have adopted a PVA-PEG hydrogel cell immobilization approach. The primary reason is to avoid liquid bacteria culture handling, ensuring both safety and user-friendliness. Another reason is to speed up the detection process. We found in the literature that hydrogel could allow hydrophobic substances, in our case, cereulide, to diffuse in the medium and better interact with biosensor cells, enhancing detection efficiency.

      To make our on-site test kits a qualified product for the market, a long enough shelf life is needed. Therefore we put spores instead of cells into the hydrogel. Sporulation of B. sub. was induced by nutrient exhaustion during construction. When the cells become endospores, they can survive in a non-favourable environment. After activation by nutrients, the performance of the sensor remains unaffected.

Figure 1 Cells to spores to gel

Hardware Cycle 0

Is PVA hydrogel good for biosensors that use fluorescence as reporters?
  • PVA powder selection
  • We purchased PVA 1799 and PVA 2699 and from the experimental trial, we chose PVA 1799 with higher molecular weight due to its better solidification and tensile strength.
  • Fluorescent Protein's Viability in Gel (Check Hardware Experiment 2)
  • We embedded GFP and RFP-expressing E. coli into gels and it shows relatively higher fluorescence colour difference by using naked eye observation on Blue Light Transilluminators.
  • Figure 2 Trapping E. coli cells in 1799 PVA Gel

    Hardware Cycle 1

    Biosignal Optimization: Hydrogel-based Cell Biosensor
    Figure 3 Scale of our Hydrogel-based Whole-cell Biosensor Disc
  • Cells: Gel Ratio (Check Hardware Experiment 2)
  • We found that the ratio of 200uL cell culture and 1300uL Gel Liquid shows viable fluorescence difference while not affecting the polymerization of the gel.
  • Size and scale of gel (Check Hardware Experiment 3)
  • We tried 12-well plates and 24-well plates for gel making.
  • 1.5 ml gel-cell mixture in a 24-well plate with a diameter of 13mm and height of 10 mm was chosen as our scale (consulted hydrogel cell culturing expert, Mr. Kachin Wong)
  • Diffusion of Hydrophobic Cereulide into the gel (Check Hardware Experiment 3)
  • Cereulide's analog chemical, Valinomycin, is dissolved in DMSO for several concentrations for testing.
  • 3uM valinomycin added to gel encapsulated spores culture gives a significant rise of Red Fluorescence Intensity after 2 hours.
  • Figure 4 Detection of Valinomycin(Analogue of Cereulide) using engineered B. Subtilis Biosensor Hydrogel

    Hardware Cycle 2 for Improving User-Friendliness and Safety

    Figure 5 Steps for Hardware Package Usage
  • Capsule Design as the container of gel biosensor (Check Hardware Prototype)
  • Sample Extraction and Applying Samples
  • How can cereulide in rice be extracted?
  • According to a research paper[1], ethanol could be used for hydrophobic cereulide extraction from rice samples, and its performance has no significant difference from methanol.
  • Will B. Subtilis Cells be killed if the solvent, ethanol, was added?
  • By plate spreading experiments (Check Hardware Experiment 1.2), we found that B. Subtilis cells are resistant to 50% ethanol solution while 75 % and 95% ethanol kill them.
  • Hardware Cycle 3 for Scalability and Storability

  • Performance stability: Cells-gel hydrogel stored for 5 days in 4°C fridge could be activated by 1 hour 37 °C incubation and able to do detection and show significant fluorescence signal (Check Hardware Experiment 3).
  • For long-term storage: Germination of spores embedded in gel (Check Hardware Experiment 3).
  • References

      [1] Delbrassinne, L., Andjelkovic, M., Rajkovic, A., Dubois, P., Nguessan, E., Mahillon, J., & Van Loco, J. (2012). Determination of Bacillus cereus Emetic Toxin in food products by means of LC-MS². Food Analytical Methods, 5, 969-979.