Purposes

In-field detection of aromatic compounds in environments has always been desirable, and convenience has always been an important requirement for in-field detection. To meet this requirement, the detection process should be fast and the result should be easily read by naked eyes. Furthermore, the device we design to realize all these should be readily portable.
When designing such a portable user-friendly device, the most challenging part would be developing the preservation method. As we choose Escherichia coli, which is unable to germinate spores, as our host strain, special method should be developed to protect the bacteria from temperature changes and physical stress while keeping them alive. We used alginate encapsulation as a basic solution to this problem and built advanced device based on such preliminary design to measure the concentration of aromatics in samples.^[5]

Alginate Encapsulation

Figure 1. The structure of alginate chelating calcium ion (a) and the beads formed after alginate was exposed to calcium ion (b).

Alginate is a polysaccharide consisting of β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues. When exposed to calcium irons, G residues will immediately cross-link to form a gel-like material that may serve as a matrix to safely contain our biosensors.
Alginate has been frequently used as the biological encapsulation material for various organisms such as Saccharomyces cerevisiae, Escherichia coli and mammalian cells^[1][2]. It stands out because of several distinct characteristics^[8]:
(1) Stable and inexpensive, even edible.
(2) Does not interfere with biosensors we constructed.
(3) Ease to shape and manipulate.
(4) Provide reliable protection against environmental stresses.
The alginate encapsulation successfully solve the problem of lethal dehydration and oxidation stress upon our biosensor strains, so no recovering process is required.

Experimental protocol:
1.5% Alginate solution was boiled and kept warm at 40°C.
E.coli was grown overnight in LB medium at 37 °C in 15 ml Falcon tube, then were harvested by centrifugation at 4000 r.p.m. for 10 minutes and then resuspended in 500 μl of fresh LB media. mixed with 3 ml alginate solution, and dropped into 0.2M calcium chloride in room temperature(20 to 24℃) to form bead-like alginate encapsulation.
Alginate beads were washed in PBS to eliminate calcium irons and then stored in the solution with protective agents or drilled-water.

Protective Agents

Inositol

Figure 2. The structure of myo-inositol. It is optically active and plays a crucial role in eukaryotic cells.

So far alginate capsule serves our purpose well. However, if additional protective agents were applied, the biosensor strains may be preserved for a longer time at 4℃, even exceeding a month.
Bacterial under dehydration may face an increasing osmotic stress as the water activity decrease. A possible way to counteract the osmotic stress is to accumulate non-toxic solutes in cytoplasm to maintain a high osmotic pressure, stablize proteins and balance the dehydration in the environment^[4]. Inositol suits this purpose well due to its stability and hydrophilicity. And this was corroborated by work done by Aitor. et al.
The link to the original paper: http://link.springer.com/article/10.1007/s00216-010-4558-y

Trehalose

Figure 3. The structure of trehalose. It is efficient in preventing dehydration.

Trehalose has an advantage over other kinds of sugars for it has a relatively high glass transition, so the bacteria may maintain a glassy form with the temperature and humidity change.Additionally, high concentration of NaCl solution induces the expression of intracellular trehalose. ^[7]

To maintain the bacteria encapsulated alive for as long as possible, the protective agents were used in high concentration. Thus, testing whether the induction process will be interfered by the protecting agents would be necessary. A series inositol and trehalose solutions with different concentration were used as medium for induction test. Results showed that the presence of protective agents does not significantly influence the behavior of our biosensors.

Figure 4. Influence of protection agents (inositol and trehalose) on the induction response of NahR biosensor. Induction ratios were measured for NahR's inducer 4-methyl-salicylic acid at 100 μM in presence of protective agents at different concentrations and compared with the induction response in presence of no protective agents (shown as the thin horizontal solid line). Results showed that these two protective agents did not interfere the inducing process of NahR biosensor even when they were in fairly high concentrations.

Results

Demonstration of our convenient in-field detection device was performed by soaking the alginate capsules containing bacteria with specific biosensors into solutions with corresponding aromatics and incubate for a certain amount of time.

Figure 5.The test results of alginate encapsulation beads with XylS and NahR biosensors in it. a: After 10h incubation, the fluorescence intensity of XylS is sufficiently high to be discriminated by blue LED and after 12 hours, by naked eyes. b:The induction experiment conducted after 12 days preservation. Left: blank. Right: response of XylS with 100μM 3-MeBzO.

For NahR biosensor, after 3 hours of incubation, the fluorescence generated by capsules soaked in solution with 100μM 4-MeBzO can be easily discerned by naked eyes using a simple blue light source, while the capsules soaked in blank solution has no visible fluorescence. The concentration of 100μM was chosen according to previous tests because under such a concentration, high induction ratio can be obtained without noticable toxicity to bacteria. (fig. 5b)

Figure 6. Tests for alginate encapsulation beads with NahR biosensor. The concentrations of inducer 4-MeSaA are: 100μM, 10μM, 1μM and blank.(Each photograph, from left to right.) and photographs were taken every hour. As is illustrated, different inducer concentration requires different induction time, and it’s possible to detect 1μM 4-MeSaA in 6 hours, this concentration is lower than the requirements by Chinese government, and the higher the concentration of inducer is, the faster detection may conduct.
(Link to the Chinese Government’s Requirements)

Figure 7. The response of NahR to the sample with 4-MeSaD treated by NahF. From left to right: 1,2: not been treated by the adaptor, NahF; 3.4: treated by NahF for 4 hours. 5,6: treated by NahF for 12 hours. 1,3,5 contain 10μM 4-MeSaD and 2,4,6 contain 100μM 4-MeSaD.

Although such demonstration was not sufficiently rigorous, it verifies the possibility that our products could be used in field to achieve a rough detection.

Advanced Design

Based previous test results on the alginate encapsulation method, we reasoned that a hydrogel patterning and transferring method could serve multi-purposes, including quantitative detection, implementing adaptors through cell communication, and realization of bandpass filter by constructing a inducer concentration gradient.

PDMS Template Design PDMS (polydimethylsiloxane) is particularly known for its unusual rheological (or flow) properties. PDMS is optically clear and, in general, inert, non-toxic, and non-flammable. PDMS is a material with no marked harmful effects on organisms in the environment and is frequently used in the microfluridic chips.
Parallel square wells which are 500μm wide, 500μm long and 170μm deep were etched on to a PDMS template, with a distance of 500 μm between them. This design was aiming at preventing interaction of E.coli between different wells. The shape of our design could be easily adjusted according to customers' need.^[5].
Pattern Transferring The alginate solution and bacterial culture mixture (described in the protocol of alginate encapsulation) was transferred into the wells. After treatment with calcium irons, the mixture solidify. The PDMS with the mixture was then transferred to a agarose layer. (Its concentration is of 1.5% or 2%) In 5 minutes, the PDMS template was peeled off with the cell patterns left on the agarose layer.
Improvements For adaptors, the adaptor E.coli cells could be cultured in the agarose layer. If the substrate of adaptor exists, it would be converted into the compound which could be dected by corresponding biosensor.
For the bandpass filter, the agarose layer could be pre-treated so that the sample and water was aliquoted on two sides of the agarose layer. After 6 to 12 hours treatment, a concentration gradient would be constructed by diffusion. (The diffusion time could be calculated according to the mass of inducers and the concentration of agarose layer.)

Figure 8. The design and experiment protocol of hydrogel patterning and transferring method. This method is potential for conducting cell communication and semi-quantitative detection.

Results

Figure 9. The primary attempt to construct the advanced device. 100μM 4-MeSaA was dropped on the left side of PDMS, then the plate was incubated in 37℃ for 6 hours. It's shown that a concentration was constructed and the response could be detected by blue LED. For the transferred patterning, This experiment also indicated that even with the PMDS adhered, the biosensor could be induced within a relative short time.

References
[1] Koch, S., Schwinger, C., Kressler, J., Heinzen, C. H., & Rainov, N. G. (2003). Alginate encapsulation of genetically engineered mammalian cells: comparison of production devices, methods and microcapsule characteristics. Journal of microencapsulation, 20(3), 303-316.
[2] Wang, N., Adams, G., Buttery, L., Falcone, F. H., & Stolnik, S. (2009). Alginate encapsulation technology supports embryonic stem cells differentiation into insulin-producing cells. Journal of biotechnology, 144(4), 304-312.
[3] Morgan, C. A., Herman, N., White, P. A., & Vesey, G. (2006). Preservation of micro-organisms by drying; a review. Journal of Microbiological Methods,66(2), 183-193.
[4] de las Heras, A., & de Lorenzo, V. (2011). In situ detection of aromatic compounds with biosensor Pseudomonas putida cells preserved and delivered to soil in water-soluble gelatin capsules. Analytical and bioanalytical chemistry, 400(4), 1093-1104.
[5] Choi, W. S., Kim, M., Park, S., Lee, S. K., & Kim, T. (2012). Patterning and transferring hydrogel-encapsulated bacterial cells for quantitative analysis of synthetically engineered genetic circuits. Biomaterials, 33(2), 624-633.
[6] Leslie, S. B., Israeli, E., Lighthart, B., Crowe, J. H., & Crowe, L. M. (1995). Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Applied and environmental microbiology, 61(10), 3592-3597.
[7] Peking iGEM 2010 wiki
[8] iGEM: Imperial Collage/Encapsulation, 2009
[9] van der Meer, J. R., & Belkin, S. (2010). Where microbiology meets microengineering: design and applications of reporter bacteria. Nature Reviews Microbiology, 8(7), 511-522.