This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial cells for use in a field portable electric cell-substrate impedance sensor. The protocol for running a rapid drinking water toxicity test with the sensor is also described.
This manuscript describes how to prepare fluidic biochips with Rainbow trout gill epithelial (RTgill-W1) cells for use in a field portable water toxicity sensor. A monolayer of RTgill-W1 cells forms on the sensing electrodes enclosed within the biochips. The biochips are then used for testing in a field portable electric cell-substrate impedance sensing (ECIS) device designed for rapid toxicity testing of drinking water. The manuscript further describes how to run a toxicity test using the prepared biochips. A control water sample and the test water sample are mixed with pre-measured powdered media and injected into separate channels of the biochip. Impedance readings from the sensing electrodes in each of the biochip channels are measured and compared by an automated statistical software program. The screen on the ECIS instrument will indicate either “Contamination Detected” or “No Contamination Detected” within an hour of sample injection. Advantages are ease of use and rapid response to a broad spectrum of inorganic and organic chemicals at concentrations that are relevant to human health concerns, as well as the long-term stability of stored biochips in a ready state for testing. Limitations are the requirement for cold storage of the biochips and limited sensitivity to cholinesterase-inhibiting pesticides. Applications for this toxicity detector are for rapid field-portable testing of drinking water supplies by Army Preventative Medicine personnel or for use at municipal water treatment facilities.
The overall goal was to develop a method for the cell seeding, storage and testing of fluidic biochips in the ECIS biosensor. The goal for the development of this biosensor was to meet US Army specifications for a field portable device that could detect possible contamination of drinking water supplies being used by soldiers. The requirements for the toxicity sensor were that it could detect a broad spectrum of toxic industrial compounds rapidly (within an hour) at concentrations relevant to human health, that the device be field-portable, and the biological components would have a shelf-life of at least nine months. Refrigeration, but not freezing, of perishable components was acceptable.
Historically, field portable water testing technologies with a biological component to them (such as antibodies, enzymes, or nucleic acids) have been analyte-specific1-3. The disadvantage to these types of biosensors is that they will only detect one type of chemical at a time. Multiple sensors are needed if it is suspected that more than one chemical is present. If a specific sensor is not in the test repertoire, chemical contaminants in the water could easily go undetected.
Broad-based toxicity sensors, on the other hand, have the potential to fill this technology gap. These usually have a cellular component to them4-8. The advantages of broad-based toxicity biosensors are that they can detect the presence of a wide array of chemical contaminants, including mixtures and unknowns, in a relatively short period of time5,9,10.
The concept of using the measurement of electrical impedance of cell monolayers as a possible toxicity sensor, which is also known as electric cell-substrate impedance sensing (ECIS), was first described by Giaever and Keese11. Over the past two decades it has been shown to be a sensitive indicator of cell viability and cytotoxicity. Basically, the cell monolayer that is adhered to the electrodes on the biochips is exposed to high frequency and low voltage and amperage alternating current signal. The confluent monolayer of cells impedes the flow of electrons. When the integrity of the cell monolayer is compromised (such as when a toxic chemical is introduced), the ECIS sensor records a change in the electrical impedance11-14. Figure 1 illustrates the principle of ECIS in relation to the cell monolayer on the biochip.
Figure 1: Principle of ECIS. Illustration of a cell monolayer on a biochip with simplified ECIS reader electrical schematic. Please click here to view a larger version of this figure.
Initially, mammalian cell lines were seeded in fluidic biochips and were used in the ECIS sensor technology described here12. These cells were not practical for field use, however, because they required frequent media changes, had a limited shelf-life, and required an artificial CO2 environment and a 37 °C incubation temperature. It was discovered that a commercially available cell line derived from rainbow trout gill epithelial cells (RTgill W-1 cells) could be tested at room temperature at ambient CO2, formed a confluent monolayer in the biochips, could be stored at refrigerated temperatures, and had a rapid response (1 hr or less) to a broad spectrum of chemicals at concentrations relevant to human health12. Applications of RTgill-W1 cells in toxicology, as well as in basic research, are reviewed by Lee et al.15
Methods for the seeding, storage and testing of fluidic biochips containing monolayers of RTgill-W1 cells on fluidic biochips in an ECIS biosensor are described here. The fluidic biochips can be stored for up to 9 months in a refrigerated state and can be shipped in a cold storage container, for testing of drinking water supplies.The accompanying ECIS readers, or test units, are shipped separately. The biochips have two components to them; an upper polycarbonate layer with two separate fluid channels, and a lower electronic layer that contains four electrode pads per channel for impedance sensing. There are 10 working electrodes per pad; each electrode is 250 µm in diameter. The assembled biochips have gold electrode connections for acquiring impedance readings when inserted into the ECIS test unit. Each of the two enclosed fluidic U-shaped channels will hold 2 ml of the RTgill-W1 cell suspension. Figure 2 shows a fluidic biochip in the ECIS reader with a magnification of a confluent cells on a single sensing electrode.
Figure 2: Fluidic Biochip in ECIS Reader. Magnified area shows a confluent monolayer of RTgill-W1 cells on a single sensing electrode. Please click here to view a larger version of this figure.
1. Preparation of Test Materials
NOTE: In order to prepare the biochips for testing, several confluent flasks of RTgill-W1 cells need to be ready. A good estimate of the number of flasks needed is one confluent T175 flask for 16 biochips to be seeded.
2. Fluidic Biochip Seeding Procedure
Note: Perform all procedures where the biochips or the media is handled in a class II biological safety cabinet using aseptic technique.
3. ECIS Testing with Biochips
Figure 3: ECIS Reader Screenshot of a Biochip with Acceptable Impedance Readings. The screenshot shows initial impedance readings in ohms of each of the 4 control electrodes (CE) and 4 test sample electrodes (SE) within the fluidic biochip. Please click here to view a larger version of this figure.
The ECIS technology described in this paper underwent testing at the US Environmental Protection Agency (USEPA)-sponsored Technology Testing and Evaluation Program (TTEP). Thirteen chemicals 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, 9 of the 13 were detected by ECIS within an hr at concentrations that are relevant to human health8. Table 1 illustrates the results of this contaminant testing. Figure 4 is representative of what a "Contaminated" result would look like on the ECIS reader screen. For the most part, cellular impedances decreased for contaminated samples as compared to controls. Occasionally, certain compounds may cause an increase in impedance.
Also summarized in Table 1 is the clean water testing. Forty clean water samples were run and no contamination was detected in any of the samples (see Figure 5) for a representative screen shot of "No Contamination Detected".
Figure 4: ECIS Reader Screenshot of a "Contaminated" Water Sample. An example of normalized impedance graphics and results from a water sample that was contaminated. Blue lines represent normalized impedances of each of the control electrodes; red lines represent normalized impedances of each of the test sample electrodes. Please click here to view a larger version of this figure.
Figure 5: ECIS Reader Screenshot of a "No Contamination Detected" Water Sample. An example of normalized impedance graphics and results from a water sample that was not contaminated. Blue lines represent normalized impedances of each of the control electrodes; red lines represent normalized impedances of each of the test sample electrodes. Please click here to view a larger version of this figure.
Category | Contaminant | Concentration Tested (mg/L)1 | Detected ≤1 hour n = 4/4 chips |
|||
Pesticides | Aldicarb | 0.17 | no | |||
Arsenic (sodium arsenite) | 4.5 | yes | ||||
Azide (sodium azide) | 46.7 | yes | ||||
Fenamiphos | 0.56 | no | ||||
Methamidophos | 1.4 | no | ||||
Methyl parathion | 33.6 | yes | ||||
Paraquat (dichloride) | 4.6 | no | ||||
Pentachlorophenate (sodium) | 71.9 | yes | ||||
Industrial Chemicals | Ammonia | 924 | yes | |||
Copper (copper II sulfate) | 103 | yes | ||||
Cyanide (sodium) | 14 | yes | ||||
Mercury (chloride) | 24.7 | yes | ||||
Toluene | 444 | yes | ||||
Clean Water2 | none | NA | no | |||
1Concentrations tested are the same as in the manuscript by Widder, et al. (2014). | ||||||
240 clean water samples were run with no contamination. |
Table 1: Contaminants in Water Samples Detected by ECIS.
The ECIS technology performed well in a laboratory setting and was able to detect potential water contaminants at concentrations that are relevant to human health. The portability and packaging of the technology makes it conducive to field use.
Critical steps in the protocol for the success of the technology are as follows: 1) Maintain aseptic conditions during culture, seeding, and feeding of the biochips, 2) Keep the seeded biochips in refrigerated conditions until ready for testing since the RTgill-W1 cells will not survive very long once they are subjected to temperatures above 25 °C, 3) Accurately weigh the L-15ex in the powdered media vials and accurately measure the water samples to avoid producing false positives, which can be caused by a shift in the osmolality of media rather than sample toxicity, 4) Follow user instructions on the ECIS screen for running the tests. The software in the reader will alert the user if a biochip is unacceptable for testing (based on initial impedance readings) when the biochip is first inserted into the reader. If the impedance levels are unacceptable for testing, the software will not allow the user to proceed with testing until a new biochip is used. Reasons for unacceptable impedance readings are usually due to a slight misalignment of the biochip electrodes with the ECIS reader pins or fluid leakage along one of the gluing edges of the biochip.
There are some limits to this technology because the ECIS sensor has only been tested with drinking water and not with surface water. The RTgill-W1 cells that are on the biochip cannot tolerate freezing or temperatures much above 25 °C for prolonged periods of time (time frame can be from hours to days dependent upon the temperature. The biochips function best in a temperature range from refrigerated to room temperature7. They are ready for immediate use, however, right after being removed from cold storage. Portable cold storage containers are currently used by Army personnel in the field for temperature-sensitive supplies. These same containers can be used for seeded biochip transport.
Another limit to this technology is that even though it is a broad-band toxicity sensor, it does not respond well, if at all, to cholinesterase-inhibiting compounds, such as some pesticides. To fill this capability gap, the ECIS sensor is designed to be used in conjunction with a commercially available rapid pesticide test assay when testing water samples in order to provide the user with a broader range of toxicity testing. The kit is a rapid enzymatic assay designed to detect organophosphate and carbamate pesticides within 30 min.
The ECIS sensor complements the WQAS-PM (Water Quality Analysis System – Preventative Medicine) field water test system, currently used by military preventative medicine personnel to detect, arsenic, lead, or cyanide in a drinking water sample. Although the ECIS sensor will not identify what the contaminant is, it will indicate if certain metals or organic compounds are present, indicating that the water may not be suitable for human consumption. The ECIS test results are available within an hr. The water samples can then be sent out for further analysis for identification of the contaminant if there is a positive test result.
As described above, the ECIS reader is designed to be part of a system that includes a separate enzymatic ACE kit in order to cover a broad spectrum of contaminant detection. Both of these readers are being packaged in a sturdy case for field transport for field use by soldiers.
The authors have nothing to disclose.
This work was supported by the US Army Medical Research and Materiel Command and by the Small Business Innovation Research and Small Business Technology Transfer program; Contract No. W81XWH-13-C-0093. We would like to thank Dr. Lucy Lee at the University of Fraser for being our RTgill-W1 cell culture mentor, and to acknowledge Dr. Niels Bols of Waterloo University for the development of the RTgill-W1cell line.
Fetal bovine serum | Life Technologies, Inc. www.lifetechnologies.com | 16000-085 | Store @ -20 °C. Thaw @ room temperature before use. Ingredient for complete L-15 cell culture media (10%). |
Fibronectin, bovine plasma | EMD Millipore Corp. www.emdmillipore.com | 341631-1 mg | Store @ -20 °C. Thaw @ room temperature before use. Mix with L-15 media for a concentration of 10 ug/mL and freeze @ -20 °C in aliquots. Use as substrate for biochips. |
L-15 media without L-glutamine | Lonza www.lonzabioscience.com | 12-700F | Basal media for cell culture and feeding biochips. Store at 6 °C. |
L-15ex powdered media with phenol red | US Biological www.usbio.net | L1501 | Media is weighed out in 60 mg aliquots in 0.1 dram vials and stored at 6 °C in foil pouches with dessicant packs. Nine month shelf-life. Mixed with 10 mL of water sample for testing in biochips. |
PBS, w/o Ca++ or Mg++ | Lonza www.lonzabioscience.com | 17-516F | Store at room temperature. Used for rinsing media when trypsinizing cell culture flasks. |
Trypsin, EDTA | Lonza www.lonzabioscience.com | CC-5012 | Store @ -20 °C. Thaw at room temperature and use to trypsinize cell culture flasks. |
T175 culture flasks | Fisher Scientific www.fishersci.com |
12-565-30 | Used for culturing RTgill-W1 cells. |
Bleach | Chlorox www.chlorox.com | Diluted to 20% with millique or distilled water for cleaning ECIS chips. Any household bleach is acceptable. | |
70 % ethyl alcohol | For disinfecting biohood surfaces and any materials being placed in biohood. | ||
Rainbow trout gill cells (RTgill-W1) | American Type Tissue Culture Collection www.atcc.org | CRL-2523 | Cells cultured and used for biosensor (seeding biochips). |
GlutaMAX-1 Supplement, 200 mM | Lonza www.lonzabioscience.com | 35050-061 | Store at room temperature. Ingredient for complete L-15 cell culture media (1%). |
Penn/Strep Stock 10K/10K | Lonza www.lonzabioscience.com | 17-602E | Store @ -20 °C. Thaw @ room temperature before use. Ingredient for complete L-15 cell culture media (1%). |
Pharmed BPT tubing | U.S. Plastic Corp. www.usplastic.com | 57317 | Cut in 27 mm sections and autoclaved. Used for seeding biochips with cells and as a closed loop between media changes. |
Polycarbonate luer fittings for Pharmed tubing assemblies | Value Plastics | MTLS210-9 | Secured to each end of cut Pharmed tubing for insertion into bichips. |
20 mL syringes, slip-tip | VWR Scientific us.vwr.com | BD302831 | Used for injection of cell suspension for seeding ECIS chips, as well as for feeding chips. |
0.1 dram snap-cap polypropylene microvials | Bottles Jars and Tubes, Inc. www.bottlesjarsandtubes.com | 30600 | Used to store 60 mg aliquots of L-15ex powdered media. |
60 mil Lexan fluidic ECIS biochips | Nanohmics, Inc. www.nanohmics.com | Custom-made by Nanohmics, Inc. RTgill-W1 cells will be injected into the biochips and seeded chips will be placed in ECIS reader for testing. | |
Autoclavable Plastic Instrument Box 17 1/2" x 7 3/4" x 2 3/8" |
Medi-Dose EPS medidose.com | IB701 | Used to store the following; autoclaved plugs, biochips that have been cleaned, seeded biochips. |
Paper heat-seal sterilization pouches, 7 ½” x 13” | CardinalHealth www.cardinalhealth.com | 90713 | Used for autoclaving tubing and fittings and plugs. |
Quantos automated powder dispenser | Mettler Toledo www.mt.com | QB5 | Automated dispension of 60 mg aliquots of powdered L-15ex into 0.1 dram vials. |
ECIS reader | Nanohmics, Inc. www.nanohmics.com | Custom-made by Nanohmics, Inc. Seeded biochip is inserted into the reader for conducting water toxicity testing. | |
3 X 5 metalized 2.5 mil polypropylene reclosable bags | Uline www.uline.com | S-16893 | Packaging and storage for both seeded biochips and powdered L-15ex media vials. |
Leatherman squirt ps4 | Amazon www.Amazon.com | Used to open powdered media vials. | |
1 gram silica gel desiccant packets | Uline www.uline.com | S-3902 | Put in polypropylene bags with L-15ex powdered media vials to prevent the powder from picking up moisture. |
Sterile 250 or 500 mL Nalgene bottles | Fisher Scientific www.fishersci.com |
09-740-25C or E | Hold cell suspensions for seeding ECIS chips in biohood. |
Plugs for biochips | Nanohmics, Inc. www.nanohmics.com | Custom-made by Nanohmics, Inc. Used to seal ports on biochips before storage @ 6°C. | |
Drains for ECIS biochips | Nanohmics, Inc. www.nanohmics.com | Custom-made by Nanohmics, Inc. Placed on 2 inner ports on biochips prior to insertion in ECIS reader. Allows for excess media to drain from channels during test injections. | |
Hemocytometer | Fisher Scientific www.fishersci.com |
S17040 | Needed for counting cells prior to adjusting cell suspension for injection into biochips. |
Brightfield microscope w/ 10X objective | Leitz Labovert | Any brightfield microscope is acceptable. | |
Class II biological safety cabinet | Any class II biological safety cabinet where cell culture can be performed under sterile conditions is acceptable. | ||
Microcentrifuge tubes, 0.6 mL | Fisher Scientific www.fishersci.com |
02-681-311 | Holds 1 mL of cell suspension prior to counting cells. |
Slip 10 cc red syringes | Procedure Products, Inc. www.procedureproducts.com | S/49S 30-R | Withdraws 9 mL of test water sample and used to inject sample into biochip. |
Slip 10 cc blue syringes | Procedure Products, Inc. www.procedureproducts.com | S/49S 30-B | Withdraws 9 mL of control water sample and used to inject sample into biochip. |
½ oz. clear pet plastic jar w/ white ribbed lined caps | SKS Bottle & Packaging, Inc. www.sks-bottle.com | 0605-30 | Sample vials used for mixing L-15ex powder and 10 mL of water sample for testing. |
50 mL sterile conical polypropylene centrifuge tubes | Fisher Scientific www.fishersci.com | 12-565-269 | Used to hold 40 mL aliquots of 10 ug/mL fibronectin @ -20 °C. |