Original Article

Design of an optical nanobiosensor for detection of Legionella pneumophila in water samples


Background and Objectives: Legionella spp. is a causative agent of Legionnaires' disease that creates public health problems. Isolation of these bacteria from water sources is essential to identify outbreak origins and prevent disease. Diagnostic biosensors for water quality control to protect consumers from water-borne infections can predict many outbreaks. Gold nanoparticles conjugated probes are a new generation of diagnostic tools. In this study, an optical nano biosensor was designed and characterized to detect Legionella pneumophila in water samples rapidly.
Materials and Methods: Thiolated probes designed for the mip gene were attached to gold nanoparticles and then water samples containing Legionella pneumophila were examined.
Results: The limit of detection for PCR and biosensor was 104 and 103 copy numbers/µl, respectively. Biosensor sensitivity and PCR were reported to be 90% (18 out of 20) and 85% (17 out of 20), respectively. Specificity 100% has been reported for both methods.
Conclusion: According to the obtained results, this method has the potential to diagnose L. pneumophila with high sensitivity and specificity. This system can be employed as a practical tool for rapid, accurate, high-sensitivity, and acceptable detection of Legionella pneumophila in contaminated water, which is cost-effective in terms of cost and time.

1. Papian S, Mohabati Mobarez A, Khoramabadi N, Mehdi Abdol M, Talebi Bezmin Abadi A. Investigating the role of L. pnuemophila LPS derivatives in formation of specific cell-mediated immune responses against the pathogen. Microb Pathog 2020; 147: 104396.
2. Grúas C, Llambi S, Arruga MV. Detection of Legionella spp. and Legionella pneumophila in water samples of Spain by specific real-time PCR. Arch Microbiol 2014; 196: 63-71.
3. Spiegelman J, Pedutem T, Francisco MJ. Legionnaires’ disease cases at a large community hospital—common and underdiagnosed. Int J Environ Res Public Health 2020; 17: 332.
4. Li J, Qin T, Jia XX, Deng AH, Zhang X, Fan WH, et al. Rapid identification of Legionella pathogenicity by surface-enhanced raman spectroscopy. Biomed Environ Sci 2015; 28: 437-444.
5. Den Boer JW, Yzerman EPF. Diagnosis of Legionella infection in Legionnaires’ disease. Eur J Clin Microbiol Infect Dis 2004; 23: 871-878.
6. Moosavian M, Moradzadeh M, Ghadiri A, Saki M. Isolation and Identification of Legionella spp. in environmental water sources based on macrophage infectivity potentiator (mip) gene sequencing in southwest Iran. AIMS Microbiol 2019; 5: 223-231.
7. Yáñez MA, Carrasco-Serrano C, Barberá VM, Catalan V. Quantitative detection of Legionella pneumophila in water samples by immunomagnetic purification and real-time PCR amplification of the dotA gene. Appl Environ Microbiol 2005; 71: 3433-3441.
8. Joly P, Falconnet P-A, André J, Weill N, Reyrolle M, Vandenesch F, et al. Quantitative real-time Legionella PCR for environmental water samples: data interpretation. Appl Environ Microbiol 2006; 72: 2801-2808.
9. Niu C, Zhang Y, Zhang Y. Evaluation of a most probable number method for detection and quantification of Legionella pneumophila. Pathogens 2022; 11: 789.
10. Islam MA, Hassen WM, Ishika I, Tayabali AF, Dubowski JJ. Selective detection of Legionella pneumophila serogroup 1 and 5 with a digital photocorrosion biosensor using antimicrobial peptide-antibody sandwich strategy. Biosensors (Basel) 2022; 12: 105.
11. Kaminker R, Lahav M, Motiei L, Vartanian M, Popovitz-Biro R, Iron MA, et al. Molecular structure–function relations of the optical properties and dimensions of gold nanoparticle assemblies. Angew Chem Int Ed Engl 2010; 49: 1218-1221.
12. Padmavathy B, Vinoth Kumar R, Jaffar Ali BM. A direct detection of Escherichia coli genomic DNA using gold nanoprobes. J Nanobiotechnology 2012; 10: 8.
13. Wang J, Lin W, Cao E, Xu X, Liang W, Zhang X. Surface plasmon resonance sensors on Raman and fluorescence spectroscopy. Sensors (Basel) 2017; 17: 2719.
14. Rasooly A, Herold KE. Biosensors and Biodetection: Methods and protocols. Preface. Methods Mol Biol 2009; 503: v-ix. 10.1007/978-1-60327-567-5.
15. Ma X, Song S, Kim S, Kwon M-S, Lee H, Park W, et al. Single gold-bridged nanoprobes for identification of single point DNA mutations. Nat Commun 2019; 10: 836.
16. Bahavarnia F, Pashazadeh-Panahi P, Hasanzadeh M, Razmi N. DNA based biosensing of Acinetobacter baumannii using nanoparticles aggregation method. Heliyon 2020; 6(7): e04474.
17. Pavlov V, Xiao Y, Shlyahovsky B, Willner I. Aptamer-functionalized Au nanoparticles for the amplified optical detection of thrombin. J Am Chem Soc 2004; 126: 11768-11769.
18. Huang C-C, Huang Y-F, Cao Z, Tan W, Chang H-T. Aptamer-modified gold nanoparticles for colorimetric determination of platelet-derived growth factors and their receptors. Anal Chem 2005; 77: 5735-5741.
19. Liu J, Lu Y. A colorimetric lead biosensor using DNAzyme-directed assembly of gold nanoparticles. J Am Chem Soc 2003; 125: 6642-6643.
20. Liu J, Lu Y. Accelerated color change of gold nanoparticles assembled by DNAzymes for simple and fast colorimetric Pb2+ detection. J Am Chem Soc 2004; 126: 12298-12305.
21. Heidari Z, Rezatofighi SE, Rastegarzadeh S. Development and comparison of cross-linking and non-crosslinking probe-gold nanoparticle hybridization assays for direct detection of unamplified bovine viral diarrhea virus-RNA. BMC Biotechnol 2021; 21: 30.
22. Wang J, Wu ZL, Zhang HZ, Li YF, Huang CZ. Selective colorimetric analysis of spermine based on the cross-linking aggregation of gold nanoparticles chain assembly. Talanta 2017; 167: 193-200.
23. Hakimian F, Ghourchian H, Hashemi AS, Arastoo MR, Behnam Rad M. Ultrasensitive optical biosensor for detection of miRNA-155 using positively charged Au nanoparticles. Sci Rep 2018; 8: 2943.
24. Wang G, Akiyama Y, Shiraishi S, Kanayama N, Takarada T, Maeda M. Cross-linking versus non-cross-linking aggregation of gold nanoparticles induced by DNA hybridization: a comparison of the rapidity of solution color change. Bioconjug Chem 2017; 28: 270-277.
25. Wang Z, Ma L. Gold nanoparticle probes. Coord Chem Rev 2009; 253: 1607- 1618.
26. Sun Y, Harris NC, Kiang C-H. Phase transition and optical properties of DNA–Gold nanoparticle assemblies. Plasmonics 2007; 2: 193-199.
27. Pal D, Boby N, Kumar S, Kaur G, Ali SA, Reboud J, et al. Visual detection of Brucella in bovine biological samples using DNA-activated gold nanoparticles. PLoS One 2017; 12(7): e0180919.28. Andreadou M, Liandris E, Gazouli M, Taka
S, Antoniou M, Theodoropoulos G, et al. A novel non-amplification assay for the detection of Leishmania spp. in clinical samples using gold nanoparticles. J Microbiol Methods 2014; 96: 56-61.
29. Kalidasan K, Neo JL, Uttamchandani M. Direct visual detection of Salmonella genomic DNA using gold nanoparticles. Mol Biosyst 2013; 9: 618-621.
30. Chen DJ, Procop GW, Vogel S, Yen-Lieberman B, Richter SS. Utility of PCR, culture, and antigen detection methods for diagnosis of legionellosis. J Clin Microbiol 2015; 53: 3474-3477.
31. Bastús NG, Comenge J, Puntes V. Kinetically controlled seeded growth synthesis of citrate-stabilized gold nanoparticles of up to 200 nm: size focusing versus Ostwald ripening. Langmuir 2011; 27: 11098-11105.
32. Karimi S, Fouani MH, Moshaii A, Nikkhah M, Hosseinkhani S, Sheikhnejad R. Development of dual functional nucleic acid delivery nanosystem for DNA induced silencing of Bcl-2 oncogene. Int J Nanomedicine 2020; 15: 1693-1708.
33. Liu X, Atwater M, Wang J, Huo Q. Extinction coefficient of gold nanoparticles with different sizes and different capping ligands. Colloids Surf B Biointerfaces 2007; 58: 3-7.
34. Wilson DA, Yen-Lieberman B, Reischl U, Gordon SM, Procop GW. Detection of Legionella pneumophila by real-time PCR for the mip gene. J Clin Microbiol 2003; 41: 3327-3330.
35. Motohashi K. A novel series of high-efficiency vectors for TA cloning and blunt-end cloning of PCR products. Sci Rep 2019; 9: 6417.
36. Nguyen TTT, Voigt K, Santiago ALCMA, Kirk PM, Lee HB. Discovery of novel Backusella (Backusellaceae, Mucorales) isolated from invertebrates and toads in Cheongyang, Korea. J Fungi (Basel) 2021; 7: 513.
37. Parikh R, Mathai A, Parikh S, Chandra Sekhar G, Thomas R. Understanding and using sensitivity, specificity and predictive values. Indian J Ophthalmol 2008; 56: 45-50.
38. Caprara D, Ripanti F, Capocefalo A, Sarra A, Brasili F, Petrillo C, et al. DNA-functionalized gold nanoparticle assemblies for Surface Enhanced Raman Scattering. Colloids Surf A Physicochem Eng Asp 2020; 589: 124399.
39. Liang P, Canoura J, Yu H, Alkhamis O, Xiao Y. Dithiothreitol-regulated coverage of oligonucleotide-modified gold nanoparticles to achieve optimized biosensor performance. ACS Appl Mater Interfaces 2018; 10: 4233-4242.
40. Collins S, Jorgensen F, Willis C, Walker J. Real‐time PCR to supplement gold‐standard culture‐based detection of Legionella in environmental samples. J Appl Microbiol 2015; 119: 1158-1169.
41. Yang G, Benson R, Pelish T, Brown E, Winchell JM, Fields B. Dual detection of Legionella pneumophila and Legionella species by real-time PCR targeting the 23S-5S rRNA gene spacer region. Clin Microbiol Infect 2010; 16: 255-261.
42. Li H, Nelson E, Pentland A, Van Buskirk J, Rothberg L. Assays based on differential adsorption of single-stranded and double-stranded DNA on unfunctionalized gold nanoparticles in a colloidal suspension. Plasmonics 2007; 2: 165-171.43.
Nuthong B, Wilailuckana C, Tavichakorntrakool R, Boonsiri P, Daduang S, Bunyaraksyotin G, et al. One step for Legionella pneumophila detection in environmental samples by DNA‐gold nanoparticle probe. J Appl Microbiol 2018; 125: 1534-1540.44. Saleh M, El-Matbouli M. Rapid detection of Cyprinid herpesvirus-3 (CyHV-3) using a gold nanoparticle-based
hybridization assay. J Virol Methods 2015; 217: 50-54.
45. Oh SY, Heo NS, Shukla S, Cho HJ, Vilian ATE, Kim J, et al. Development of gold nanoparticle-aptamer-based LSPR sensing chips for the rapid detection of Salmonella typhimurium in pork meat. Sci Rep 2017; 7: 10130.
46. Wang Y, Alocilja EC. Gold nanoparticle-labeled biosensor for rapid and sensitive detection of bacterial pathogens. J Biol Eng 2015; 9: 16.
47. Suaifan GA, Alhogail S, Zourob M. Rapid and low-cost biosensor for the detection of Staphylococcus aureus. Biosens Bioelectron 2017; 90: 230-237.
48. Wen J, Liu J, Wu J, He D. Rapid measurement of waterborne bacterial viability based on difunctional gold nanoprobe. RSC Adv 2022; 12: 1675-1681.
49. Liandris E, Gazouli M, Andreadou M, Čomor M, Abazovic N, Sechi LA, et al. Direct detection of unamplified DNA from pathogenic mycobacteria using DNA-derivatized gold nanoparticles. J Microbiol Methods 2009; 78: 260-264.
IssueVol 14 No 6 (2022) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijm.v14i6.11254
Biosensor; Probe; Legionella pneumophila; Water; Nanoparticles

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
Karimiravesh R, Mohabati Mobarez A, Behmanesh M, Nikkhah M, Talebi Bezmin Abadi A, Esmaeilli S. Design of an optical nanobiosensor for detection of Legionella pneumophila in water samples. Iran J Microbiol. 2022;14(6):802-812.