The overall goal of this procedure is to demonstrate how to use a biosensor to evaluate the toxicity of drinking water samples. This is accomplished by creating biochips which contain a monolayer of rainbow trout gill cells and then using them as part of an impedence-based biosensor. This procedure provides a way to rapidly detect chemical toxicity in drinking water supplies at levels that are relevant to human health.

The main advantages of this technique are that the biochips can be used under field conditions, they can be stored cold for up to nine months without any care or maintenance of the cells, and that they are at a ready state for testing and can be used at any point after removal from refrigeration. Demonstrating this procedure will be Jordan McNairn, an undergraduate student from our laboratory. The day before cell seeding, remove the biochips from their packaging and place them into sterilized plastic instrument cases.

Attach one of the tubing assemblies to the syringe and draw up a 20%bleach solution into a 20 milliliter syringe. Inject two milliliters of the solution into each channel of the biochip. When finished, allow the biochips to sit for one hour with the bleach solution.

After one hour, use a sterile one-milliliter syringe fitted with a male slip luer assembly attached to a vacuum line to aspirate the bleach solution from both channels. After aspirating the bleach from the biochip, attach a sterile tubing assembly with male slip luers at each end to the biochip to use as a drain while rinsing the biochips. Then, draw up sterile water into a 20 milliliter syringe, and flush each channel of the chip with five milliliters of water.

Vacuum aspirate the water as before. And place the biochips back into the plastic instrument cases. Leave the biochips in the bio-safety hood until the next day.

One hour prior to seeding the biochips, inject two milliliters of a 10 micrograms per milliliter fibronectin solution into each channel of the biochip. Incubate the biochips in the bio-safety hood for 60 minutes, and then vacuum aspirate the fibronectin solution. Finally, place two sterile biochip tubing assemblies onto the ports of the biochips, and set the biochips aside until the cells are ready for seeding.

Remove one confluent T-175 flask containing rainbow trout gill epithelial cells from the incubator, and place it into the bio-safety hood. One confluent T-175 flask will be able to seed approximately 16 biochips. Aspirate off the media from the cells.

Rinse them with PBS, and aspirate off PBS. And then add six milliliters of Trypsin-EDTA solution to the cell layer. Incubate the flask at room temperature for about five minutes.

Then, add 15 milliliters of complete L-15 cell culture media to each flask, to deactivate the trypsin. Take a small aliquot from the cell suspension and use a hemocytometer to measure the concentration of the cells. View the cells using a bright field microscope equipped with a 10x objective.

Then, calculate the current cell concentration and adjust the cell suspension to a concentration of a quarter of a million cells per milliliter. Next, use a sterile 20 milliliter syringe to inject 2.5 milliliters of the cell suspension into the outer port of each channel of the biochip. Allow the extra cell suspension to flow out of the tubing into a waste container.

Then create an enclosed loop for each biochip channel by inserting the free end of the hose with a luer fitting into the outer port for each channel. Next, moisten a paper towel with 70%ethanol and use it to wipe any excess media off of the closed loops. Then, place the biochips back in a plastic box.

On days four and seven, remove the biochips from the incubator. And change the media in the biochips with temperature-equilibrated complete L-15 cell culture media. After the day seven feeding, remove and discard the hoses from the chips and insert the autoclave drain plugs into the biochips.

Place the biochips in a box in a six degree Celsius incubator until they are used for testing. Remove the ECIS test reader and supplies from the carrying case, and then turn on the reader. Also, take out one of the prepared biochips from the six degree Celsius incubator and place it onto a paper towel.

Using a 10 milliliter syringe, dispense 10 milliliters of control water into a labeled 0.5-ounce clear plastic control jar. Be sure to remove bubbles for an accurate measurement. Then, use a separate syringe and dispense 10 milliliters of the test sample into a 0.5 ounce clear plastic test jar.

Next, remove two powdered media vials. Open one of the powdered media vials and pour the entire contents of one vial into the jar with the control water, dropping the entire vial into solution as well. Repeat this procedure with the test jar.

Then, cap and shake the jars to ensure that the powder is completely dissolved. Fill the blue 10 milliliter syringe with nine milliliters of the control solution. And the red 10 milliliter syringe with nine milliliters of the test solution from their respective vials.

Remove any existing air bubbles from the syringes. Next, remove the plugs from the biochip ports and attach the drain. Place the biochip into the removable plastic tray in the ECIS reader, close the lid, and then attach the filled syringes to the outer biochip ports.

Finally, attach the syringe plungers. From the main screen of the reader, select Next to initiate the pre-test. The software will then check the initial impedances.

If the impedances are within range, the screen will register Cartridge passed"and instruct the user to select Next. Then, enter sample information using a soft keyboard, and then select Accept when finished. At this point, two minutes of impedance data will be collected from the inserted biochip, and an onscreen timer will count down time.

After two minutes, hit Next. When prompted by a flashing green box to Inject samples now"inject the control and test media from the attached syringes simultaneously into the biochip channels. Leave the syringes in place on the biochips when finished.

If the ECIS reader software determines that the treatment channel is statistically different from the control channel at any point during the test, the onscreen display will indicate that the sample is contaminated. Otherwise it will indicate that no contamination is detected at the end of the monitoring period. Record test results as Contaminated"or Not contaminated"for each sample.

At the end of the test run, remove and discard the biochip. Rinse and air-dry the syringes, the test vials and the removable plastic tray which housed the biochip during the tests. 13 chemicals, shown here, were selected for testing as representatives of a broad spectrum of toxic industrial compounds that could be possible contaminants of drinking water.

During the testing, nine of the 13 were detected by electric cell substrate impedance sensing within an hour, at concentrations that are relevant to human health. Typical outputs that would be seen following an electric cell substrate impedance sensing test are shown here. Contaminated samples end up with significantly lower impedance levels, when compared to the control, clean water samples.

After watching this video, you should have a good understanding of how to prepare a biochip with a monolayer of rainbow trout gill epithelial cells and to subsequently use that biochip in an impedance-based toxicity test for the evaluation of drinking water toxicity. While attempting this procedure, it's important to remember to work aseptically in preparing the biochips, and also to keep the biochips refrigerated until they are used. Each biochip can only be used once and then must be discarded.

Once mastered, the biochip preparation can be done in about 10 minutes. This is an estimate, because chips are prepared in batches. Sample impedance testing, including sample preparation takes about one hour and 10 minutes if it's performed properly.

Following this procedure, other methods such as analytical chemistry can be performed on a sample that produced a contaminated result, in order to identify the suspected chemical or chemicals. Development of this technique paves the way for researchers interested in water toxicity sensing to utilize technology using rainbow trout gill epithelial cells and impedance sensing. We hope that you will find the techniques useful and look forward to future innovations that these new capabilities may help to facilitate.

Thank you and have a great day.

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