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.
总体目标是开发用于细胞接种,储存和流体生物芯片的测试中的ECIS生物传感器的方法。对于该生物传感器的开发目标是,以满足美国陆军规格,可以检测饮用水供应的可能的污染正在由士兵一个字段便携式装置。对于毒性传感器的要求是,它可以在有关对人类健康浓度迅速检测有毒工业化合物的广谱(一个小时内),该装置是现场便携式,和生物组分将具有一个货架寿命至少九个月。冷藏,但不冻结,易腐成分是可以接受的。
从历史上看,与生物成分将它们现场便携式水检测技术(如抗体,酶,或核酸)已经被分析物特异性1-3。这些类型的生物传感器的缺点在于,它们将ONLŸ同时检测一种类型的化学品。如果怀疑该多个化学是本需要多个传感器。如果特定的传感器是不是在测试曲目,在水中化学污染物很容易被忽视。
广泛的毒性传感器,另一方面,必须填写此项技术差距的潜力。这些通常具有细胞成分对他们4-8。广泛的毒性生物传感器的优点是它们可以检测各种各样的化学污染物,其中包括它们的混合物和未知数,在时间5,9,10相对较短的存在。
使用细胞单层的电阻抗作为一个可能的毒性传感器,它也被称为电细胞-基底阻抗感测(ECIS)的测量的概念,首先由Giaever和Keese酒店11所述。在过去的二十年中,已经证明是细胞VIAB的敏感指标ility和细胞毒性。基本上,即附着在电极上的生物芯片的细胞单层暴露于高频率和低的电压和电流强度交流电流信号。细胞的汇合单层阻碍电子的流动。当细胞单层的完整性受到损害(当被引入一种有毒化学物质,如),ECIS的传感器记录在电阻抗的变化11-14。 图1示出ECIS的原则关系到细胞单层上的生物芯片。
图1:关于与简化ECIS读者电气原理生物芯片细胞单层ECIS插图原理 请点击此处查看该图的放大版本。
对于播种,存储和容纳流体生物芯片检测方法就在ECIS传感器流体生物芯片RTgill-W1细胞单层如下所述。流体生物芯片可以保存长达9个月在冷藏状态,并且可以在一个冷藏集装箱运,对于饮用水supplies.The伴随ECIS阅读器,或测试单元的测试,被单独提供。生物芯片有两个组成部分的人;上部的聚碳酸酯层,用两个分开的流体通道,以及包含每第四频道电极焊盘用于阻抗传感低级电子层。有每个垫10工作电极;每个电极是250微米的直径。组装的生物芯片具有用于当插入ECIS测试单元获取阻抗读数金电极连接。每两个封闭流体的U形通道将持有2毫升RTgill-W1细胞悬浮液; 图2示出了在ECIS读者汇合的细胞上的单个感测电极的倍率的流体的生物芯片。
<p class=“jove_content”FO:保together.within页=“1”>ECIS的技术在实验室环境下表现良好,并能在浓度是相关的人体健康检测潜在的水污染物。该技术的便携和包装使得它有利于现场使用。
在该协议的技术成功的关键步骤如下:1)培养,播种期间保持无菌条件下,与生物芯片的馈送,2)保持种子生物芯片在冷藏条件下,直到准备用于自RTgill-W1细胞测试很长,一旦它们进行温度高于25℃时,将无法生存,3)准确称L形15ex在粉状介质小瓶和精确地测量水样品,以避免产生假阳性,其可通过在移位引起的媒体而不是样品毒性渗透压,4)按照ECIS屏幕上的用户说明运行测试。在阅读器软件将提醒如果双向用户ochip是用于测试(基于初始阻抗读数)不可接受时生物芯片首先插入读卡器。如果阻抗水平测试不能接受的,该软件将不允许用户与测试继续进行,直到一个新的生物芯片时使用。为不可接受的阻抗读数的原因通常是由于与ECIS读者销或沿着所述生物芯片的胶合边缘之一流体泄漏的生物芯片的电极的轻微未对准。
有一些限制这种技术,因为ECIS传感器只被用饮用水测试和不与表面水。所述RTgill-W1细胞是在生物芯片上不能较长时间(时间帧可以是从数小时至数天取决于温度耐受冷冻或温度大大高于25℃。在生物芯片起作用的温度范围最好从冷冻以室温7,他们准备立即使用,然而,作为后R权从冷库emoved。便携式冷藏容器目前在该领域使用的军队人员对温度敏感的用品。这些相同的容器可以用于种子生物芯片运输。
这种技术的另一个限制是,即使它是一个宽波段毒性传感器,它不,如果在所有反应良好,以抑制胆碱酯酶的化合物,如某些杀虫剂。为了填补这一能力差距,ECIS的传感器被设计以提供具有更广泛的毒性测试的用户测试水样时,在结合使用市售的快速农药试验测定。该试剂盒是一种快速酶法测定设计为在30分钟内检测有机磷和氨基甲酸酯杀虫剂。
ECIS的传感器补充WQAS-PM(水质分析系统 – 预防医学)域水测试系统,目前使用的军事预防医学人员发现,阿尔森IC,铅,氰化物或饮用水样本。虽然ECIS传感器将不能识别污染物是什么,这将表明,如果某些金属或有机化合物的存在,这表明水可能不适合人食用。 ECIS的测试结果可用一个小时之内。然后将水样可发出用于进一步分析的污染物的鉴定是否有阳性测试结果。
如上所述,ECIS的读者被设计为,其包括为了覆盖污染物检测的广谱单独酶ACE试剂盒的系统的一部分。这两个读者都被包装在运输领域为士兵野外使用一个坚固的情况下。
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. |