Efficient separation and sensitive identification of pathogenic bacterial strains is essential for a prosperous modern society, with direct applications in medical diagnostics, drug discovery, biodefense, and food safety. We developed a fast and reliable method for antibody-based selective immobilization of bacteria from suspension onto a gold-plated glass surface, followed by detection using strain-specific antibodies linked to gold nanoparticles decorated with a reporter molecule. The reporter molecules are subsequently detected by surface-enhanced Raman spectroscopy (SERS). Such a multi-functionalized nanoparticle is called a SERS-tag. The presented procedure uses widely accessible and cheap materials for manufacturing and functionalization of the nanoparticles and the immobilization surfaces. Here, we exemplify the use of the produced SERS-tags for sensitive single-cell detection of opportunistic pathogen Escherichia coli, and we demonstrate the selectivity of our method using two other bacterial strains, Staphylococcus aureus and Serratia marcescens, as negative controls. We believe that the described approach has a potential to inspire the development of novel medical diagnostic tools for rapid identification of bacterial pathogens.

Department of Microbiology Faculty of Medicine of Masaryk University and St Anne's University Hospital Pekařská 53 656 91 Brno Czech Republic

Institute of Scientific Instruments of the Czech Academy of Sciences v v i Královopolská 147 612 64 Brno Czech Republic

Zobrazit více v PubMed

Nikaido H. Multidrug Resistance in Bacteria. Annu. Rev. Biochem. 2009;78:119–146. doi: 10.1146/annurev.biochem.78.082907.145923. PubMed DOI PMC

French K., Evans J., Tanner H., Gossain S., Hussain A. The Clinical Impact of Rapid, Direct MALDI-ToF Identification of Bacteria from Positive Blood Cultures. PLoS ONE. 2016;11:e0169332. doi: 10.1371/journal.pone.0169332. PubMed DOI PMC

Ruiz-Aragón J., Ballestero-Téllez M., Gutiérrez-Gutiérrez B., de Cueto M., Rodríguez-Baño J., Pascual Á. Direct Bacterial Identification from Positive Blood Cultures Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry: A Systematic Review and Meta-Analysis. Enfermedades Infecc. Microbiol. Clin. Engl. Ed. 2018;36:484–492. doi: 10.1016/j.eimc.2017.08.012. PubMed DOI

Ahmed A., Rushworth J.V., Hirst N.A., Millner P.A. Biosensors for Whole-Cell Bacterial Detection. Clin. Microbiol. Rev. 2014;27:631–646. doi: 10.1128/CMR.00120-13. PubMed DOI PMC

Samek O., Bernatová S., Dohnal F. The Potential of SERS as an AST Methodology in Clinical Settings. Nanophotonics. 2021;10:2537–2561. doi: 10.1515/nanoph-2021-0095. DOI

Dudak F.C., Boyacı İ.H. Rapid and Label-Free Bacteria Detection by Surface Plasmon Resonance (SPR) Biosensors. Biotechnol. J. 2009;4:1003–1011. doi: 10.1002/biot.200800316. PubMed DOI

Temur E., Boyacı İ.H., Tamer U., Unsal H., Aydogan N. A Highly Sensitive Detection Platform Based on Surface-Enhanced Raman Scattering for Escherichia Coli Enumeration. Anal. Bioanal. Chem. 2010;397:1595–1604. doi: 10.1007/s00216-010-3676-x. PubMed DOI

Maquelin K., Kirschner C., Choo-Smith L.-P., van den Braak N., Endtz H.P., Naumann D., Puppels G.J. Identification of Medically Relevant Microorganisms by Vibrational Spectroscopy. J. Microbiol. Methods. 2002;51:255–271. doi: 10.1016/S0167-7012(02)00127-6. PubMed DOI

Bernatová S., Samek O., Pilát Z., Šerý M., Ježek J., Jákl P., Šiler M., Krzyžánek V., Zemánek P., Holá V., et al. Following the Mechanisms of Bacteriostatic versus Bactericidal Action Using Raman Spectroscopy. Molecules. 2013;18:13188–13199. doi: 10.3390/molecules181113188. PubMed DOI PMC

Pilát Z., Bernatová S., Ježek J., Kirchhoff J., Tannert A., Neugebauer U., Samek O., Zemánek P. Microfluidic Cultivation and Laser Tweezers Raman Spectroscopy of E. Coli under Antibiotic Stress. Sensors. 2018;18:1623. doi: 10.3390/s18051623. PubMed DOI PMC

Rebrošová K., Šiler M., Samek O., Růžička F., Bernatová S., Ježek J., Zemánek P., Holá V. Differentiation between Staphylococcus Aureus and Staphylococcus Epidermidis Strains Using Raman Spectroscopy. Future Microbiol. 2017;12:881–890. doi: 10.2217/fmb-2016-0224. PubMed DOI

Bernatová S., Rebrošová K., Pilát Z., Šerý M., Gjevik A., Samek O., Ježek J., Šiler M., Kizovský M., Klementová T., et al. Rapid Detection of Antibiotic Sensitivity of Staphylococcus Aureus by Raman Tweezers. Eur. Phys. J. Plus. 2021;136:233. doi: 10.1140/epjp/s13360-021-01152-1. DOI

Hakonen A., Svedendahl M., Ogier R., Yang Z.-J., Lodewijks K., Verre R., Shegai T., Andersson P.O., Käll M. Dimer-on-Mirror SERS Substrates with Attogram Sensitivity Fabricated by Colloidal Lithography. Nanoscale. 2015;7:9405–9410. doi: 10.1039/C5NR01654A. PubMed DOI

Woo M.-A., Lee S.-M., Kim G., Baek J., Noh M.S., Kim J.E., Park S.J., Minai-Tehrani A., Park S.-C., Seo Y.T., et al. Multiplex Immunoassay Using Fluorescent-Surface Enhanced Raman Spectroscopic Dots for the Detection of Bronchioalveolar Stem Cells in Murine Lung. Anal. Chem. 2009;81:1008–1015. doi: 10.1021/ac802037x. PubMed DOI

Lin C.-C., Yang Y.-M., Chen Y.-F., Yang T.-S., Chang H.-C. A New Protein A Assay Based on Raman Reporter Labeled Immunogold Nanoparticles. Biosens. Bioelectron. 2008;24:178–183. doi: 10.1016/j.bios.2008.03.035. PubMed DOI

Vo-Dinh T., Yan F., Stokes D.L. Plasmonics-Based Nanostructures for Surface-Enhanced Raman Scattering Bioanalysis. In: Vo-Dinh T., editor. Protein Nanotechnology: Protocols, Instrumentation, and Applications. Humana Press; Totowa, NJ, USA: 2005. pp. 255–283. Methods in Molecular BiologyTM. PubMed

Chan W.C.W., Nie S. Quantum Dot Bioconjugates for Ultrasensitive Nonisotopic Detection. Science. 1998;281:2016–2018. doi: 10.1126/science.281.5385.2016. PubMed DOI

Li Y., Wang Z., Mu X., Ma A., Guo S. Raman Tags: Novel Optical Probes for Intracellular Sensing and Imaging. Biotechnol. Adv. 2017;35:168–177. doi: 10.1016/j.biotechadv.2016.12.004. PubMed DOI

Shrivastav A.M., Cvelbar U., Abdulhalim I. A Comprehensive Review on Plasmonic-Based Biosensors Used in Viral Diagnostics. Commun. Biol. 2021;4:70. doi: 10.1038/s42003-020-01615-8. PubMed DOI PMC

Kim K., Kashefi-Kheyrabadi L., Joung Y., Kim K., Dang H., Chavan S.G., Lee M.-H., Choo J. Recent Advances in Sensitive Surface-Enhanced Raman Scattering-Based Lateral Flow Assay Platforms for Point-of-Care Diagnostics of Infectious Diseases. Sens. Actuators B Chem. 2021;329:129214. doi: 10.1016/j.snb.2020.129214. PubMed DOI PMC

Jarvis R.M., Brooker A., Goodacre R. Surface-Enhanced Raman Scattering for the Rapid Discrimination of Bacteria. Faraday Discuss. 2006;132:281–292. doi: 10.1039/B506413A. PubMed DOI

Kitahama Y., Itoh T., Pienpinijtham P., Ekgasit S., Han X.X., Ozaki Y. Biological Applications of SERS Using Functional Nanoparticles. In: Hepel M., Zhong C.-J., editors. ACS Symposium Series. Vol. 1113. American Chemical Society; Washington, DC, USA: 2012. pp. 181–234.

Kearns H., Goodacre R., Jamieson L.E., Graham D., Faulds K. SERS Detection of Multiple Antimicrobial-Resistant Pathogens Using Nanosensors. Anal. Chem. 2017;89:12666–12673. doi: 10.1021/acs.analchem.7b02653. PubMed DOI

Guven B., Basaran-Akgul N., Temur E., Tamer U., Boyacı İ.H. SERS-Based Sandwich Immunoassay Using Antibody Coated Magnetic Nanoparticles for Escherichia Coli Enumeration. The Analyst. 2011;136:740–748. doi: 10.1039/C0AN00473A. PubMed DOI

Huang K., Martí A.A. Recent Trends in Molecular Beacon Design and Applications. Anal. Bioanal. Chem. 2012;402:3091–3102. doi: 10.1007/s00216-011-5570-6. PubMed DOI

Heydari E. Nanoplasmonic Biodetection Based on Bright-Field Imaging of Resonantly Coupled Gold-Silver Nanoparticles. Photonics Nanostructures. 2019;36:100708. doi: 10.1016/j.photonics.2019.100708. DOI

Liu J., He H., Xiao D., Yin S., Ji W., Jiang S., Luo D., Wang B., Liu Y. Recent Advances of Plasmonic Nanoparticles and Their Applications. Materials. 2018;11:1833. doi: 10.3390/ma11101833. PubMed DOI PMC

Ma Y., Cai F., Li Y., Chen J., Han F., Lin W. A Review of the Application of Nanoparticles in the Diagnosis and Treatment of Chronic Kidney Disease. Bioact. Mater. 2020;5:732–743. doi: 10.1016/j.bioactmat.2020.05.002. PubMed DOI PMC

Fu X., Cai J., Zhang X., Li W.-D., Ge H., Hu Y. Top-down Fabrication of Shape-Controlled, Monodisperse Nanoparticles for Biomedical Applications. Adv. Drug Deliv. Rev. 2018;132:169–187. doi: 10.1016/j.addr.2018.07.006. PubMed DOI

Wang A.X., Kong X. Review of Recent Progress of Plasmonic Materials and Nano-Structures for Surface-Enhanced Raman Scattering. Mater. Basel Switz. 2015;8:3024–3052. doi: 10.3390/ma8063024. PubMed DOI PMC

Rong Z., Wang C., Wang J., Wang D., Xiao R., Wang S. Magnetic Immunoassay for Cancer Biomarker Detection Based on Surface-Enhanced Resonance Raman Scattering from Coupled Plasmonic Nanostructures. Biosens. Bioelectron. 2016;84:15–21. doi: 10.1016/j.bios.2016.04.006. PubMed DOI

Han X.X., Kitahama Y., Itoh T., Wang C.X., Zhao B., Ozaki Y. Protein-Mediated Sandwich Strategy for Surface-Enhanced Raman Scattering: Application to Versatile Protein Detection. Anal. Chem. 2009;81:3350–3355. doi: 10.1021/ac802553a. PubMed DOI

Güçlü K., Özyürek M., Güngör N., Baki S., Apak R. Selective Optical Sensing of Biothiols with Ellman’s Reagent: 5,5′-Dithio-Bis(2-Nitrobenzoic Acid)-Modified Gold Nanoparticles. Anal. Chim. Acta. 2013;794:90–98. doi: 10.1016/j.aca.2013.07.041. PubMed DOI

Baniukevic J., Hakki Boyaci I., Goktug Bozkurt A., Tamer U., Ramanavicius A., Ramanaviciene A. Magnetic Gold Nanoparticles in SERS-Based Sandwich Immunoassay for Antigen Detection by Well Oriented Antibodies. Biosens. Bioelectron. 2013;43:281–288. doi: 10.1016/j.bios.2012.12.014. PubMed DOI

Tamer U., Boyacı İ.H., Temur E., Zengin A., Dincer İ., Elerman Y. Fabrication of Magnetic Gold Nanorod Particles for Immunomagnetic Separation and SERS Application. J. Nanoparticle Res. 2011;13:3167–3176. doi: 10.1007/s11051-010-0213-y. DOI

Brandt N.N., Brovko O.O., Chikishev A.Y., Paraschuk O.D. Optimization of the Rolling-Circle Filter for Raman Background Subtraction. Appl. Spectrosc. 2006;60:288–293. doi: 10.1366/000370206776342553. PubMed DOI

Salehi M., Mittelstaedt W., Packeisen J., Haase M., Hamann A. An Alternative Way to Prepare Biocompatible Nanotags with Increased Reproducibility of Results. J. Nanomater. Mol. Nanotechnol. 2016;5:1000181. doi: 10.4172/2324-8777.1000181. DOI

Le Ru E.C., Blackie E., Meyer M., Etchegoin P.G. Surface Enhanced Raman Scattering Enhancement Factors: A Comprehensive Study. J. Phys. Chem. C. 2007;111:13794–13803. doi: 10.1021/jp0687908. DOI

Bakshi S., Leoncini E., Baker C., Cañas-Duarte S.J., Okumus B., Paulsson J. Tracking Bacterial Lineages in Complex and Dynamic Environments with Applications for Growth Control and Persistence. Nat. Microbiol. 2021;6:783–791. doi: 10.1038/s41564-021-00900-4. PubMed DOI PMC

Lin Y.-C., Huang C., Lai H.-C. Revealing the Ultrastructure of the Membrane Pores of Intact Serratia Marcescens Cells by Atomic Force Microscopy. Heliyon. 2019;5:e02636. doi: 10.1016/j.heliyon.2019.e02636. PubMed DOI PMC

Jorge A.M., Hoiczyk E., Gomes J.P., Pinho M.G. EzrA Contributes to the Regulation of Cell Size in Staphylococcus Aureus. PLoS ONE. 2011;6:e27542. doi: 10.1371/journal.pone.0027542. PubMed DOI PMC