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. a,The structure of alginate chelating calcium ion.b,the beads formed after alginate was exposed to calcium ion.

Alginate is a polysaccharide consisting of β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues. When exposed to calcium ions, 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 ions and then stored in the solution with protective agents or drilled-water.

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 time.

We adopted NahR biosensor for the low basal level and high induction ratio. Based on previous works determining the most suitable concentration range for induction, our device was exposed to inducers below 100 μM, because it is found that higher concentration of inducer may inhibit the bacteria's growth.

4 different inducer concentrations were tested which are selected according to previous works and environmental standards. We tracked the change of fluorescence intensity in six hours. Every hour the photographs of 4 concentrations were taken respectively. It is obvious that this device is capable enough to meet the national environmental standards for aromatics, as well as it is user friendly and efficiency.(Figure.6)

Figure 6. Tests for alginate encapsulation beads with NahR biosensor. Vertical line represents concentrations of inducer 4-MeSaA. They are 0μM, 1μM, 10μM and 100μM respectively. Horizontal line stands for time points in six hours. As is illustrated, 6 hours is sufficient for our device to detect 1μM 4-MeSaA, which is lower than the requirements by Chinese government, with naked eyes.
(Link to the Chinese Government’s Requirements)

To further combine with Adptors to expand detection profile, this device coating NahR was cultured in LB medium in which Adptor NahF had been treated previously. Evident fluorescence could also be observed, indicating that it is possible to connect this device with adaptors. But it still needs further research to confirm.

All the demonstration above has verified that our products are usable. Comparing with traditional chemical methods which need laboratory work, it is convenient to operate. As for sensitivity, though this device couldn't compare with chemical methods, it is capable enough to meet environmental standards for aromatics. So it could be used in field to achieve a rough detection of aromatics.

Advanced Design

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

PDMS Template Design PDMS (polydimethylsiloxane), particularly known for its unusual rheological (or flow) properties, is optically clear and, in general, inert, non-toxic, and non-flammable. It 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 pattern of our design could be easily adjusted according to customers' need.^[5].
Alginate Pattern formation Alginate is still adopted as encapsulation. The mixture of alginate and bacterial culture was added into the wells of PDMS. After treating with calcium ions, the mixture solidify. Then PDMS with the mixture was adhered upside-down to an agarose layer whose concentration is of 1.5% or 2%. After 5 minutes incubation in 40℃, the PDMS template was peeled off with the solidified alginate mixture left on the agarose layer, forming corresponding pattern.

Figure 8. The design and experiment protocol of hydrogel patterning and transferring method. This method can be applied to 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.

For further improvement, this device could be combined with adaptors, the adaptor E.coli cells could be cultured in agarose layer. When exposed to suitable substrates, it would be converted into compounds which is be detected by corresponding biosensor located in alginate.
For combination with the Band-pass filter, the bacterial strains possessing different detecting profiles as well as different concentration ranges could be encapsulated into alginate and located in distinct units on agarose. When treated with unknown samples, reporter gene sfGFP shows different fluorescence intensity in each strain, thus it is possible to deduce types and concentrations of certain aromatics in the samples.

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.