Purposes

To realize the idea of in-field detection, a device with remarkable convenience for monitoring environmental water pollution should be proposed. This device must be capable of determining if the specific kind of aromatic compound exists in a water sample, and perhaps more significantly, measuring specific aromatic compound’s concentration semi-quantitatively. To meet the requirement of convenience, the detection process must be fast and the result must be read with naked eyes or with user-friendly devices. As for measuring the concentration, a concentration gradient could be constructed by the pre-treating method of the device, so different response patterns may roughly reflect the concentration. All this requirements must be carefully designed with biosafety concerns.
The most challenging part of a biological detection device is the preservation method. The sensor strain we use, Escherichia coli, failed to germinate spores or gemma to resist general preservation conditions of dehydration, temperature changes and physical interference. Several approaches were designed to achieve valid maintainance benefits. Based on the fundamental designs, an advanced device with multifunctions was proposed to measure the concentration carrying potential for further improvements.

Alginate Encapsulation

Figure 1. The structure of alginate and the cross-link between encapsulation product.

Alginate is a polysaccharide consists of β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues. If treated by calcium irons, G residues are cross-linked and a coat would be formed within a rather short period of time.
Alginate is frequently used as the biological encapsulation material for various organisms such as Saccharomyces cerevisiae, Escherichia coli and mammalian cells[1][2]. It came into our consideration as the encapsulating material for its outstanding specifications, which include[8]:
(1) Food grade materials which are stable and inexpensive
(2) Possibility to use every biosensor we constructed
(3) Ease to shape and manipulate
(4) Protection against environmental stresses
The alginate encapsulation successfully solve the problem of dehydration and oxidation stress upon our biosensor strains, so no reviving process is required. With the support of protective agents, the period of validity could be longer than a month in 4℃.

Experimental protocol:
1.5% Alginate solution was boiled and kept warm in 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, which is the active steroisomer, having crucial function in eukaryotic cells.

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 compatible solutes to maintain a high osmotic pressure in cytoplasm to stablize proteins and balance the dehydration in the environment[4]. Inositol is highly compatible for this usage for its stability and hydrophilicity.
Previous work done by Aitor. et al. showed that inositol is highly efficient for preserving bacterial cells. (In that case, P. putida, which is the host of the transcriptional factors we used in our biosensors. )

Figure 3. The results indicated that inositol was a protective agent of maintained the viability of bacterial cells. With the existance of maltodextrins(MD), the optimal validity maintaining could be achieved. (figure from: A. Heras and V. Lorenzo, 2011)
The link to the original paper: http://link.springer.com/article/10.1007/s00216-010-4558-y

Trehalose

Figure 4.The structure of trehalose, which is a disarrcharide that is a major constituent of insects surving as energy storage compound. It has also been found 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. It is also important to produce in cell trehalose by the bacteria spontaneously, which involves certain gene expression and an osmotic press induction. In E. coli, the otsA and otsB genes are responsible for trehalose biosynthesis from UDP glucose. These genes encode trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, respectively. And high concentration of NaCl solution induces the expression of intracellular trehalose. ^[7]
To obtain a high validity maintainance, the protective agents’ we use were of ralatively high concentration, so we should test that the induction process was not interfered by the protecting agents. A series inositol and trehalose solutions of different concentration were tested.

Figure 5.The tests of induction with the existance of different concentration of protective agents, inositol and trehalose. The NahR was induced by 100μM of inducers. As is illustrated, these two protective agents didn’t interfere the inducing process within a fairly high concentration.

Advanced Designs

Based on the alginate encapsulation method and previous test results, a hydrogel patterning and transferring method could surve our purposes. This method would be multi-purposes including aromatic detection in a quantitative view, possibility of the cell communication serving the idea of adaptor, and potential for the application 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, which is frequently used in the microfluridic chips.
The parallel wells were etched on PDMS, using a PDMS template in which 500 μm×500 μm square patterns are microfabricated and equidistant from each other by 500 μm. The depth of the wells was 170μm. This design was aiming at prevent interaction of E.coli between different suqares, because the interaction may influence the diffusion process for constucting a concentration gradient. Addtionally, the shape could be altered according to the basal level and detection convenience^[5].
Pattern Transferring The alginate solution and bacterial culture mixture(described in the protocol of alginate encapsulation) was transferred in the wells. After treated with Calcium irons, the PDMS with the mixture was 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 substrate.
Improvements For adaptors(Link:), the adaptor E.coli cells could be encapsulated in the agarose layer. Then if the substrate of adaptor exist, it would be tranferred into the inducer which could be dected by corresponding biosensor. For the bandpass filter(Link:), the agarose layer could be pre-treated, that sample and water was drilled on each side 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 6. The design and experiment protocol of hydrogel patterning and transferring method. This method is potential for conducting cell communication and semi-quantitative detection.

Figure 7.

Figure 8.