Optimizing Dual Fluorescent Analysis to Investigate the Toxicity of AgNPs in E. coli

Wei Hong1, Shaopeng Chen1

1 Key Laboratory of Environmental Toxicology and Pollution Control Technology of Anhui Province, Hefei, Anhui
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 20.14
DOI:  10.1002/cptx.28
Online Posting Date:  August, 2017
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The ever‐increasing use of silver nanoparticles (AgNPs) carries potential ecotoxicological risks. For full risk assessment, E. coli cells harboring a plasmid with a constitutively expressed GFP gene under control of lac promoter (lac::GFP) are extensively utilized. Flow cytometry is an advanced technology usually applied to toxicological research for rapid, efficient, multi‐parameter analysis of single cells. However, it is difficult to accurately and sensitively detect the toxicity of nanoparticles with flow cytometry due to the interference of aggregated nanoparticles. In this protocol, dual‐fluorescence detection with a propidium iodide (PI)–lac::GFP assay is used to determine the toxicity of AgNPs and successfully discriminate the dead or fragilized bacteria from living bacteria and aggregated nanoparticles. © 2017 by John Wiley & Sons, Inc.

Keywords: silver nanoparticles; toxic effects; dual fluorescence analysis; flow cytometry

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Characterization of AgNPs in Test Media
  • Support Protocol 1: Pre‐Incubating AgNPs in 2× Test Medium
  • Basic Protocol 2: Colony‐Forming Units Experiment
  • Support Protocol 2: Preparation of Bacteria Stock
  • Basic Protocol 3: Flow Cytometry Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Characterization of AgNPs in Test Media

  Materials
  • AgNPs (40‐nm, Sigma Aldrich)
  • 2× test media: distilled deionized H 2O, 2× mLB medium (see recipe), and 0.4× mLB medium (see recipe)
  • 96‐well microplates (Corning Costar)
  • Orbital shaker (Zhicheng, cat. no. ZWY‐200D)
  • High‐resolution transmission electron microscope (HRTEM, JEOL JEM‐2010)
  • 96‐well plates (Corning Costar)
  • Microplate reader (SpectraMax M2; Molecular Devices)
  • Additional reagents and equipment for pre‐incubating AgNPs in test medium ( protocol 2) and transmission electron microscopy (Burghardt & Droleskey, )

Support Protocol 1: Pre‐Incubating AgNPs in 2× Test Medium

  Materials
  • AgNPs (40‐nm, Sigma Aldrich)
  • 2× test media: distilled deionized H 2O, 2× mLB medium (see recipe), and 0.4× mLB medium (see recipe)
  • 15‐ml test tubes (e.g., Corning Falcon)
  • Orbital shaker (Zhicheng, cat. no. ZWY‐200D)

Basic Protocol 2: Colony‐Forming Units Experiment

  Materials
  • LB medium (see recipe)
  • Bacteria stock (see protocol 4)
  • 2× test media: distilled deionized H 2O, 2× mLB medium (see recipe), and 0.4× mLB medium (see recipe)
  • AgNPs (40‐nm, Sigma Aldrich)
  • LB agar plates with 100 µg/ml ampicillin
  • 15‐ml test tubes (e.g., Corning Falcon)
  • Orbital shaker (Zhicheng, cat. no. ZWY‐200D)
  • 96‐well plates (Corning Costar)
  • Additional reagents and equipment for pre‐incubating AgNPs in test medium ( protocol 2)

Support Protocol 2: Preparation of Bacteria Stock

  Materials
  • E. coli DH5α competent cells
  • pUC18‐GFP plasmid DNA (constructed by inserting GFP into EcoRI‐SalI‐digested pUC18 plasmid (Takara, cat. no. 3218))
  • SOC medium (see recipe)
  • LB agar plates with 100 µg/ml ampicillin (see recipe)
  • 50% glycerol (see recipe)
  • LB medium (see recipe)
  • 1.5 ml microcentrifuge tubes
  • 42° shaking water bath
  • 15‐ml tubes (e.g., Corning Falcon)
  • Orbital shaker (Zhicheng, cat. no. ZWY‐200D)

Basic Protocol 3: Flow Cytometry Analysis

  Materials
  • LB medium (see recipe)
  • Bacteria stock (see protocol 4)
  • 0.2× mLB (see recipe)
  • AgNPs (10 nm, 40 nm, 100 nm, Sigma‐Aldrich)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 1 mg/ml propidium iodide (PI; BIODEE, cat.no DE0301)
  • E. coli DH5α competent cells
  • Silver ions (silver nitrate; Sigma‐Aldrich, cat. no. 204390)
  • 15‐ml tubes (e.g., Corning Falcon)
  • Refrigerated centrifuge
  • 96‐well plates (Corning Costar)
  • Orbital shaker (Zhicheng, cat. no. ZWY‐200D)
  • 5‐ml round‐bottom flow cyometry tubes (Corning Falcon, cat. no. 352052)
  • Flow cytometer (BD FACSCalibur)
  • BD CELLQuest software
  • FlowJO 7.6.1 software
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
  Beddow, J., Stolpe, B., Cole, P., Lead, J. R., Sapp, M., Lyons, B. P., … Whitby, C. (2014). Effects of engineered silver nanoparticles on the growth and activity of ecologically important microbes. Environmental Microbiology Reports, 6, 448–458. doi: 10.1111/1758‐2229.12147.
  Belkin, S. (2003). Microbial whole‐cell sensing systems of environmental pollutants. Current Opinion in Microbiology, 6, 206–212. doi: 10.1016/S1369‐5274(03)00059‐6.
  Burghardt, R. C., & Droleskey, R. (2006). Transmission electron microscopy. Current Protocols in Microbiology, 3, 2B.1.1–2B.1.39. doi: 10.1002/9780471729259.mc02b01s03.
  Chambers, B. A., Afrooz, A. R., Bae, S., Aich, N., Katz, L., Saleh, N. B., & Kirisits, M. J. (2014). Effects of chloride and ionic strength on physical morphology, dissolution, and bacterial toxicity of silver nanoparticles. Environmental Science & Technology, 48(1)761–769. doi: 10.1021/es403969x.
  Chen, M., Yang, Z., Wu, H., Pan, X., Xie, X., & Wu, C. (2011). Antimicrobial activity and the mechanism of silver nanoparticle thermosensitive gel. International Journal of Nanomedicine, 6, 2873–2877. doi: 10.2147/IJN.S23945.
  Gao, J., Powers, K., Wang, Y., Zhou, H., Roberts, S. M., Moudgil, B. M., … Barber, D. S. (2012). Influence of Suwannee River humic acid on particle properties and toxicity of silver nanoparticles. Chemosphere, 89, 96–101. doi: 10.1016/j.chemosphere.2012.04.024.
  Gogoi, S. K., Gopinath, P., Paul, A., Ramesh, A., Ghosh, S. S., & Chattopadhyay, A. (2006). Green fluorescent protein‐expressing Escherichia coli as a model system for investigating the antimicrobial activities of silver nanoparticles. Langmuir, 22, 9322–9328. doi: 10.1021/la060661v.
  Hadrup, N., & Lam, H. R. (2014). Oral toxicity of silver ions, silver nanoparticles and colloidal silver—A review. Regulatory Toxicology and Pharmacology, 68, 1–7. doi: 10.1016/j.yrtph.2013.11.002.
  Hwang, E. T., Lee, J. H., Chae, Y. J., Kim, Y. S., Kim, B. C., Sang, B. I., & Gu, M. B. (2008). Analysis of the toxic mode of action of silver nanoparticles using stress‐specific bioluminescent bacteria. Small, 4, 746–750. doi: 10.1002/smll.200700954.
  Inada, T., Kimata, K., & Aiba, H. (1996). Mechanism responsible for glucose‐lactose diauxie in Escherichia coli: Challenge to the cAMP model. Genes to Cells, 1, 293–301. doi: 10.1046/j.1365‐2443.1996.24025.x.
  Jaiswal, J. K., & Simon, S. M. (2015). Imaging live cells using quantum dots. Cold Spring Harbor Protocols, 2015(7):619‐625. doi: 10.1101/pdb.top086322.
  Jung, W. K., Koo, H. C., Kim, K. W., Shin, S., Kim, S. H., & Park, Y. H. (2008). Antibacterial activity and mechanism of action of the silver ion in Staphylococcus aureus and Escherichia coli. Applied and Environmental Microbiology, 74, 2171–2178. doi: 10.1128/AEM.02001‐07.
  Lehtinen, J., Nuutila, J., & Lilius, E. M. (2004). Green fluorescent protein‐propidium iodide (GFP‐PI) based assay for flow cytometric measurement of bacterial viability. Cytometry. Part A, 60, 165–172. doi: 10.1002/cyto.a.20026.
  Li, F., Lei, C., Shen, Q., Li, L., Wang, M., Guo, M., … Yao, S. (2013). Analysis of copper nanoparticles toxicity based on a stress‐responsive bacterial biosensor array. Nanoscale, 5, 653–662. doi: 10.1039/C2NR32156D.
  Li, X., & Lenhart, J. J. (2012). Aggregation and dissolution of silver nanoparticles in natural surface water. Environmental Science & Technology, 46, 5378–5386. doi: 10.1021/es204531y.
  Li, W.‐R., Xie, X.‐B., Shi, Q.‐S., Zeng, H.‐Y., OU‐Yang, Y.‐S., & Chen, Y.‐B. (2009). Antibacterial activity and mechanism of silver nanoparticles on Escherichia coli. Applied Microbiology and Biotechnology, 85, 1115–1122. doi: 10.1007/s00253‐009‐2159‐5.
  Liang, X., Soupir, M. L., Rigby, S., Jarboe, L. R., & Zhang, W. (2014). Flow cytometry is a promising and rapid method for differentiating between freely suspended Escherichia coli and E. coli attached to clay particles. Journal of Applied Microbiology, 117, 1730–1739. doi: 10.1111/jam.12660.
  Liu, H. L., Dai, S. A., Fu, K. Y., & Hsu, S. H. (2010). Antibacterial properties of silver nanoparticles in three different sizes and their nanocomposites with a new waterborne polyurethane. International Journal of Nanomedicine, 5, 1017–1028. doi: 10.2147/IJN.S14572.
  Lok, C. N., Ho, C. M., Chen, R., He, Q. Y., Yu, W. Y., Sun, H., … Che, C. M. (2006). Proteomic analysis of the mode of antibacterial action of silver nanoparticles. Journal of Proteome Research, 5, 916–924. doi: 10.1021/pr0504079.
  Reinsch, B. C., Levard, C., Li, Z., Ma, R., Wise, A., Gregory, K. B., … Lowry, G. V. (2012). Sulfidation of silver nanoparticles decreases Escherichia coli growth inhibition. Environmental Science & Technology, 46, 6992–7000. doi: 10.1021/es203732x.
  Sahni, G., Gopinath, P., & Jeevanandam, P. (2013). A novel thermal decomposition approach to synthesize hydroxyapatite‐silver nanocomposites and their antibacterial action against GFP‐expressing antibiotic resistant E. coli. Colloids and Surfaces. B, Biointerfaces, 103, 441–447. doi: 10.1016/j.colsurfb.2012.10.050.
  Si, Y., Grazon, C., Clavier, G., Rieger, J., Audibert, J. F., Sclavi, B., & Meallet‐Renault, R. (2015). Rapid and accurate detection of Escherichia coli growth by fluorescent pH‐sensitive organic nanoparticles for high‐throughput screening applications. Biosensors & Bioelectronics, 75, 320–327. doi: 10.1016/j.bios.2015.08.028.
  Tejamaya, M., Romer, I., Merrifield, R. C., & Lead, J. R. (2012). Stability of citrate, PVP, and PEG coated silver nanoparticles in ecotoxicology media. Environmental Science & Technology, 46, 7011–7017. doi: 10.1021/es2038596.
  Yagur‐Kroll, S., Bilic, B., & Belkin, S. (2010). Strategies for enhancing bioluminescent bacterial sensor performance by promoter region manipulation. Bioengineered Bugs, 1, 151–153. doi: 10.4161/bbug.1.2.11104.
  Yin, Y., Yang, X., Zhou, X., Wang, W., Yu, S., Liu, J., & Jiang, G. (2015). Water chemistry controlled aggregation and photo‐transformation of silver nanoparticles in environmental waters. Journal of Environmental Sciences, 34, 116–125. doi: 10.1016/j.jes.2015.04.005.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library