Abstract
Understanding the mechanism of nanoparticle (NP) induced toxicity in microbes is of potential importance to a variety of disciplines including disease diagnostics, biomedical implants, and environmental analysis. In this context, toxicity to bacterial cells and inhibition of biofilm formation by GaN NPs and their functional derivatives have been investigated against gram positive and gram negative bacterial species down to single cellular level. High levels of inhibition of biofilm formation (>80 %) was observed on treatments with GaN NPs at sub-micro molar concentrations. These results were substantiated with morphological features investigated with field emission scanning electron microscope, and the observed changes in vibrational modes of microbial cells using Raman spectroscopy. Raman spectra provided molecular interpretation of cell damage by registering signatures of molecular vibrations of individual living microbial cells and mapping the interplay of proteins at the cell membrane. As compared to the untreated cells, Raman spectra of NP-treated cells showed an increase in the intensities of characteristic protein bands, which confirmed membrane damage and subsequent release of cellular contents outside the cells. Raman spectral mapping at single cellular level can facilitate understanding of the mechanistic aspect of toxicity of GaN NPs. The effect may be correlated to passive diffusion causing mechanical damage to the membrane or ingress of Ga3+ (ionic radius ~0.076 nm) which can potentially interfere with bacterial metabolism, as it resembles Fe2+ (ionic radius ~0.077 nm), which is essential for energy metabolism.
Similar content being viewed by others
References
Abedini F, Hosseinkhani H, Ismail M, Domb AJ, Omar AR, Chong PP, Hong PD, Yu DS, Farber IY (2012) Cationized dextran nanoparticle-encapsulated CXCR4-siRNA enhanced correlation between CXCR4 expression and serum alkaline phosphatase in a mouse model of colorectal cancer. Int J Nanomed 7:4159–4168
Anselme K, Davidson P, Popa AM, Giazzon M, Liley M, Ploux L (2010) The interaction of cells and bacteria with surfaces structured at the nanometre scale. Acta Biomater 6:3824–3846
Auffan M, Rose J, Wiesner MR, Bottero J-Y (2009) Chemical stability of metallic nanoparticles: a parameter controlling their potential cellular toxicity in vitro. Environ Pollut 157:1127–1133
Bordi C, de Bentzmann S (2011) Hacking into bacterial biofilms: a new therapeutic challenge. Ann Intensive care 1:19
Chan JW, Winhold H, Corzett MH, Ulloa JM, Cosman M, Balhorn R, Huser T (2007) Monitoring dynamic protein expression in living E. coli. Bacterial cells by laser tweezers Raman spectroscopy. Cytometry A 71A:468–474
Chapman J, Wier E, Regan F (2010) Period four metal nanoparticles on the inhibition of biofouling. Colloids Surf B 78:208–216
Chen L, Wen Y-M (2011) The role of bacterial biofilm in persistent infections and control strategies. Int J Oral Sci 3:66–73
Chitambar CR, Matthaeus WG, Antholine WE, Graff K, O’Brien WJ (1988) Inhibition of leukemic HL60 cell growth by transferrin-gallium: effects on ribonucleotide reductase and demonstration of drug synergy with hydroxyurea. Blood 72:1930–1936
Choi O, Deng KK, Kim M-J, Ross L, Surampalli RY, Hu Z (2008) The inhibitory effects of silver nanoparticles silver ions and silver chloride colloids on microbial growth. Water Res 42:3066–3074
Choi O, Yu C-P, Fernandez GE, Hu Z (2010) Interactions of nanosilver with Escherichia coli cells in planktonic and biofilm cultures. Water Res 44:6095–6103
Choo-Smith LP, Van Maquelin K, Vreeswijk T, Bruining HA, Puppels GJ, Ngo Thi NA, Kirschner C, Naumann D, Ami D, Villa AM et al (2001) Investigating microbial (micro)colony heterogeneity by vibrational spectroscopy. Appl Environ Microbiol 67:1461–1469
Coenye T, Nelis HJ (2010) In vitro and in vivo model systems to study microbial biofilm formation. J Micro Methods 83:89–105
Flemming H-C, Wingender J (2010) The biofilm matrix. Nat Rev Microbiol 8:623–633
Gil PR, Oberdorster G, Elder A, Puntes V, Parak WJ (2010) Correlating physico-chemical with toxicological properties of nanoparticles: the present and the future. ACS Nano 4:5527–5531
Haka AS, Volynskaya Z, Gardecki JA, Nazemi J, Lyons J, Hicks D, Fitzmaurice M, Dasari RR, Crowe JP, Field MS (2006) In vivo margin assessment during partial mastectomy breast surgery using Raman spectroscopy. Cancer Res 66:3317–3322
Hall-Stoodley L, Costerton JW, Stoodley P (2004) Bacterial biofilms: from the Natural environment to infectious diseases. Nat Rev Microbiol 2:95–108
Harz M, Rosch P, Peschke K-D, Ronneberger O, Burkhardt H, Popp J (2005) Micro-Raman spectroscopic identification of bacterial cells of the genus Staphylococcus and dependence on their cultivation conditions. Analyst 130:1543–1550
Hassett DJ (1993) Cloning and characterization of the Pseudomonas aeruginosa sodA and sodB genes encoding manganese- and iron-cofactored superoxide dismutase: demonstration of increased manganese superoxide dismutase activity in alginate-producing bacteria. J Bacteriol 175:7658–7665
Hequet A, Humblot V, Berjeaud J, Pradier C (2011) Optimized grafting of antimicrobial peptides on stainless steel surface and biofilm resistance tests. Colloids Surf B 84:301–309
Hoiby N, Ciofu O, Johansen KH, Song Z, Moser C, Jensen PO, Molin S, Givskov M, Tolker-Nielsen T, Bjarnsholt T (2011) The clinical impact of bacterial biofilms. Int J Oral Sci 3:55–65
Hosseinkhani H, Hosseinkhani M, Gabrielson NP, Pack DW, Khademhosseini A, Kobayashi H (2008) DNA nanoparticles encapsulated in 3D tissue-engineered scaffolds enhance osteogenic differentiation of mesenchymal stem cells. J Biomed Mater Res A 85A:47–60
Ivleva NP, Wagner M, Szkola A, Horn H, Niessner R, Haisch C (2010) Label-free in situ SERS imaging of biofilms. J Phys Chem B 114:10184–10194
Jewett SA, Makowski MS, Andrews B, Manfra MJ, Ivanisevic A (2012) Gallium nitride is biocompatible and non-toxic before and after functionalization with peptides. Acta Biomater 8:728–733
Kaneko Y, Thoendel M, Olakanmi O, Britigan BE, Singh PK (2007) The transition metal gallium disrupts Pseudomonas aeruginosa iron metabolism and has antimicrobial and antibiofilm activity. J Clin Invest 117:877–888
Krafft C, Knetschke T, Siegner A, Funk RHW, Salzer R (2003) Mapping of single cells by near infrared Raman microspectroscopy. Vib Spectrosc 32:75–83
Liu S, Wei L, Hao L, Fang N, Chang MW, Xu R, Yang Y, Chen Y (2009) Sharper and faster “nano darts” kill more bacteria: a study of antibacterial activity of individually dispersed pristine single-walled carbon nanotube. ACS Nano 3:3891–3902
Liu R, Liu J, Zhou X, Jiang G (2011) Application of Raman-based techniques to on-site and in vivo analysis. Trends Anal Chem 30:1462–1476
Liu X, Chen G, Keller AA, Su C (2013) Effects of dominant material properties on the stability and transport of TiO2 nanoparticles and carbon nanotubes in aquatic environments: from synthesis to fate. Environ Sci Process Impacts 15:169–189
Mahmoudi M, Hosseinkhani H, Hosseinkhani M, Boutry S, Simchi A, Journeay WS, Subramani K, Laurent S (2011) Magnetic resonance imaging tracking of stem cells in vivo using iron oxide nanoparticles as a tool for the advancement of clinical regenerative medicine. Chem Rev 111:253–280
Monteiro DR, Gorup LF, Takamiya AS, Ruvollo-Filho AC, Rodrigues de Camargo E, Barbosa DB (2009) The growing importance of materials that prevent microbial adhesion: antimicrobial effect of medical devices containing silver. Int J Antimicrob Agents 34:103–110
Murthy PS, Venkatesan R (2009) Industrial biofilms and their control. In: Flemming HC, Murthy PS, Venkatesan R, Cooksey K (eds) Marine and industrial biofouling, Springer series on biofilms. Springer, Heidelberg, pp 65–103
Murthy PS, Sahoo P, Venugopalan VP, Dhara S, Saini G, Das A, Tyagi AK (2011) Gallium oxide nanoparticle induced inhibition of bacterial adhesion and biofilm formation. IEEE 2011:490–493. doi:10.1109/ICONSET.2011.6168010
Narasimhan J, Ancholine WE, Chitambar CR (1992) Effect of gallium on the tyrosyl radical of the iron-dependent M2 subunit of ribonucleotide reductase. Biochem Pharmacol 44:2403–2408
Nel AE, Madler L, Velegol D, Xia T, Hoek EMV, Somasundaran P, Klaessig F, Castranova V, Thompson M (2009) Understanding biophysicochemical interactions at the nano–bio interface. Nat Mater 8:543–557
Niemeyer CM (2001) Nanoparticles, proteins, and nucleic acids: biotechnology meets materials science. Angew Chem Int Ed 40:4128–4158
Olakanmi O, Britigan BE, Schlesinger LS (2000) Gallium disrupts iron metabolism of mycobacteria residing within human macrophages. Infect Immun 68:5619–5627
Premasiri WR, Mori DT, Klempner MS, Krieger N, Joness G II, Ziegler LD (2005) Characterization of the surface enhanced Raman scattering of bacteria. J Phys Chem B109:312–320
Rogan MP, Taggart CC, Greene CM, Murphy PG, ONeill SJ, Mc Elvaney NG (2004) Loss of microbicidal activity and increased formation of biofilm due to decreased lactoferrin activity in patients with cystic fibrosis. J Infect Dis 190:1245–1253
Rosch P, Harz M, Schmitt M, Peschke K, Ronneberger O, Burkhardt H, Motzkus H, Lankers M, Hofer S, Thiele H et al (2005) Chemotaxonomic identification of single bacteria by micro-Raman spectroscopy: application to clean room relevant biological contaminations. Appl Environ Microbiol 71:1626–1637
Ruparelia PJ, Chatterjee AK, Duttagupta SP, Mukherji S (2008) Strain specificity in antimicrobial activity of silver and copper nanoparticles. Acta Biomater 4:707–716
Saar BG, Zeng YN, Freudiger CW, Liu YS, Hummel ME, Xie XS, Ding SY (2010) Label-free real-time monitoring of biomass processing with stimulated Raman scattering microscopy. Angew Chem Int Ed Engl 49:5476–5479
Sahoo P, Dhara S, Das CR, Dash S, Tyagi AK, Raj B, Chandramohan P, Srinivasan MP (2010) Surface optical modes in GaN nanowires. Int J Nanotechnol 7:823–832
Sahoo P, Dhara S, Dash S, Tyagi AK (2011) One dimensional GaN nanostructures: growth kinetics and applications. Nanosci Nanotechnol Asia 2:140–170
Sahoo P, Dhara S, Amirthapandian S, Kamruddin M, Dash S, Tyagi AK (2012) Role of surface polarity in self-catalyzed nucleation and evolution of GaN nanostructures. Cryst Growth Des 2:2375–2381
Sahoo P, Sumathi S, Dhara S, Saini G, Rangarajan S, Tyagi AK (2013) Direct label free ultrasensitive impedimetric DNA biosensor using dendrimer functionalized GaN nanowires. Biosens Bioelectron 15:164–170
Sandt C, Smith TP, Pink J, Brennan L, Pink D (2007) Confocal Raman microspectroscopy as a tool for studying the chemical heterogeneities of biofilms in situ. J Appl Microbiol 103:1808–1820
Schuster KC, Urlaub E, Gapes JR (2000) Single-cell analysis of bacteria by Raman microscopy: spectral information on the chemical composition of cells and on the heterogeneity in a culture. J Microbiol Methods 42:29–38
Siel JR, Webster TJ (2011) Reduced Staphylococcus aureus proliferation and biofilm formation on zinc oxide nanoparticle PVC composite surfaces. Acta Biomater 7:2579–2584
Simoes M, Simoes LC, Vieira MJ (2010) A review of current and emergent biofilm control strategies. LWT Food Sci 43:573–583
Singamaneni S, Gupta M, Yang R, Tomczak MM, Naik RR, Wang ZL, Tsukruk VV (2009) Nondestructive in situ identification of crystal orientation of anisotropic ZnO nanostructures. ACS Nano 3:2593–2600
Singh PK, Parsek MR, Greenberg EP, Welsh MJ (2002) A component of innate immunity prevents bacterial biofilm development. Nature 417:552–555
Sinha R, Karan R, Sinha A, Khare SK (2011) Interaction and nanotoxic effect of ZnO and Ag nanoparticles on mesophilic and halophilic bacterial cells. Bioresour Technol 102:1516–1522
Socrates G (2001) Infrared and Raman characteristic group frequencies: tables and charts. Wiley, Chichester. ISBN:0-470-09307-2
Sondi I, Salopek-Sondi B (2004) Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria. Colloid Interface Sci 275:177–182
Stephen M (2008) Life as a nanoscale phenomenon. Angew Chem Int Ed 47:5306–5320
Subramani K, Hosseinkhani H, Khraisat A, Hosseinkhani M, Pathak Y (2009) Targeting nanoparticles as drug delivery systems for cancer treatment. Curr Nanosci 5:135–140
Subramani K, Pathakb S, Hosseinkhani H (2012) Recent trends in diabetes treatment using nanotechnology. Dig J Nanomaterials Biostructures 7:85–95
Takahashi A, Yomoda S, Ushijima Y, Kobayashi I, Inuoue M (1995) Ofloxacin norfloxacin and ceftazidime increase the production of alginate and promote the formation of biofilm of Pseudomonas aeruginosa in vitro. J Antimicrob Chemother 36:743–745
Valappil SP, Ready D, Abou EA, Pickup DM, O’Dell LA, Chrzanowski W, Pratten J, Newport RJ, Smith ME, Wilson M et al (2009) Controlled delivery of antimicrobial gallium ions from phosphate-based glasses. Acta Biomater 5:1198–1210
Vitol EA, Orynbayeva Z, Bouchard MJ, Azizkhan-Clifford J, Friedman G, Gogotsi Y (2009) In situ intracellular spectroscopy with surface enhanced Raman spectroscopy-enabled nanopipettes. ACS Nano 3:3529–3536
Wagner V, Dullaart A, Bock AK, Zweck A (2006) The emerging nanomedicine landscape. Nat Biotechnol 24:1211–1217
Wagner M, Ivleva NP, Haisch C, Niessner R, Horn H (2009) Combined use of confocal laser scanning microscopy and Raman microscopy: investigations on EPS-Matrix. Water Res 43:63–76
Wingender J, Flemming HC (2011) Biofilms in drinking water and their role as reservoir for pathogens. Int J Hyg Environ Health 214:417–423
Yan GH, Wang GJ, Li YC (1991) Effects of alpha-dimethylamino-cyclohexoxyl-dimethyl gallium on ultrastructure of erythrocytic stage of Plasmodium berghei and P yoelii. Acta Pharmacol Sin 12:530–533
Yoon K, Byeon JH, Park J, Hwang J (2007) Susceptibility constants of Escherichia coli and Bacillus subtilis to silver and copper nanoparticles. Sci Total Environ 373:572–575
Zhang L, Jiang Y, Ding Y, Daskalakis N, Jeuken L, Povey M, O’Neill AJ, York DW (2010) Mechanistic investigation into antibacterial behaviour of suspensions of ZnO nanoparticles against E. coli. J Nanopart Res 12:1625–1636
Zhang H, Ji Z, Xia T, Meng H, Low-Kam C, Liu R, Pokhrel S, Lin S, Wang X, Liao YP, Wang M, Li L, Rallo R, Damoiseaux R, Telesca D, Mädler L, Cohen Y, Zink JI, Nel AE (2012) Use of metal oxide nanoparticle band gap to develop a predictive paradigm for oxidative stress and acute pulmonary inflammation. ACS Nano 273:4349–4368
Acknowledgments
We thank M. Kamurudin, SND for FESEM analysis, and S. Dash, SND, IGCAR for general support and encouragement.
Author information
Authors and Affiliations
Corresponding authors
Additional information
Prasana Sahoo and P. Sriyutha Murthy have contributed equally to this work.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Sahoo, P., Murthy, P.S., Dhara, S. et al. Probing the cellular damage in bacteria induced by GaN nanoparticles using confocal laser Raman spectroscopy. J Nanopart Res 15, 1841 (2013). https://doi.org/10.1007/s11051-013-1841-9
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s11051-013-1841-9