Journal of Solid State Electrochemistry

, Volume 22, Issue 5, pp 1321–1330 | Cite as

Optimization and characterization of a biosensor assembly for detection of Salmonella Typhimurium

  • A. M. A. Melo
  • D. L. Alexandre
  • M. R. F. Oliveira
  • R. F. Furtado
  • M. F. Borges
  • P. R. V. Ribeiro
  • A. Biswas
  • H. N. Cheng
  • C. R. Alves
  • E. A. T. Figueiredo
Original Paper


The performance of biosensors depends directly on the strategies adopted during their development. In this paper, a fast and sensitive biosensor for Salmonella Typhimurium detection was assembled by using optimization studies in separate stages. The pre-treatment assays, biomolecular immobilization (primary antibody and protein A concentrations), and analytical response (hydroquinone and hydrogen peroxide concentrations) were optimized via voltammetric methods. In the biosensor assembly, a gold surface was modified via the self-assembled monolayer technique (SAM) using cysteamine thiol and protein A for immobilization of anti-Salmonella antibody. The analytical response of the biosensor was obtained through the use of a secondary antibody labeled with a peroxidase enzyme, and the signal was evaluated by applying the chronoamperometry technique. The biosensor was characterized by infrared spectroscopy and cyclic voltammetry. Optimization of protein A and primary antibody concentrations enabled higher analytical signals of 7.5 and 75 mg mL−1, respectively, to be achieved. The hydroquinone and H2O2 concentrations selected were 3 and 300 mM, respectively. The biosensor developed attained a very low detection limit of 10 CFU mL−1 and a fast response with a final detection time of 125 min. These results indicate that this biosensor is very promising for the food safety and emergency response applications.


Salmonella Immunosensor Amperometric Pathogen Rapid detection 



The authors would like to thank the Brazilian agencies, CNPq, FUNCAP, and CAPES, for their financial support, Embrapa Tropical Agroindustry and National Center of Energy and Materials Research (CNPEM). Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.


  1. 1.
    Centers for disease control and prevention - CDC (2012) Pathogens causing US foodborne illnesses, hospitalizations, and deaths, 2000–2008 Accessed 10 Apr 2017
  2. 2.
    Andrews WH, Wang H, Jacobson A, Hammack TS (2016) Salmonella. In: FOOD AND DRUG ADMINISTRATION. Bacteriological analytical manual (BAM) on line. Chap. 5.>. Accessed 12 Apr 2017
  3. 3.
    Velusamy V, Arshak K, Korostynska O, Oliwa K, Adley C (2010) An overview of foodborne pathogen detection: in the perspective of biosensors. Biotechnol Adv 28:232–254CrossRefGoogle Scholar
  4. 4.
    Gil ES, Melo GR (2010) Electrochemical biosensors in pharmaceutical analysis. Braz J Pharm Sci 46:375–381CrossRefGoogle Scholar
  5. 5.
    Arora P, Sindhu A, Kaur H, Dilbaghi N, Chaudhury A (2013) An overview of transducers as platform for the rapid detection of foodborne pathogens. Appl Microbiol Biotechnol 97:1829–1836CrossRefGoogle Scholar
  6. 6.
    Wang Y, Duncan TV (2017) Nanoscale sensors for assuring the safety of food products. Curr Opin Biotechnol 44:74–86CrossRefGoogle Scholar
  7. 7.
    Kokkinos C, Economou A, Prodromidis MI (2016) Electrochemical immunosensors: critical survey of different architectures and transduction strategies. Trac Trends Anal Chem 79:88–105CrossRefGoogle Scholar
  8. 8.
    Skladal P (1997) Advances in electrochemical immunosensors. Electroanalysis 9(10):737–745CrossRefGoogle Scholar
  9. 9.
    Skladal P, Kovar D, Krajicek V, Siskova P, Pribyl J, Svabenska E (2013) Electrochemical immunosensors for detection of microorganisms. Int J Electrochem Sci 8(2):1635–1649Google Scholar
  10. 10.
    Ricci F, Adornetto G, Palleschi G (2012) A review of experimental aspects of electrochemical immunosensors. Electrochim Acta 84:74–83CrossRefGoogle Scholar
  11. 11.
    Carvalhal RF, Kubota LT, Freire RS (2005) Polycrystalline gold electrodes: a comparative study of pretreatment procedures used for cleaning and thiol self-assembly monolayer formation. Electroanalysis 17(14):1251–1259CrossRefGoogle Scholar
  12. 12.
    Pimenta-Martins MGR, Furtado RF, Heneine LGD, Dias RS, Borges MD, Alves CR (2012) Development of an amperometric immunosensor for detection of staphylococcal enterotoxin type a in cheese. J Microbiol Methods 91:138–143CrossRefGoogle Scholar
  13. 13.
    Salam F, Tothill IE (2009) Detection of Salmonella typhimurium using an electrochemical immunosensor. Biosens Bioelectron 24(8):2630–2636CrossRefGoogle Scholar
  14. 14.
    Babacan S, Pivarnik P, Letcher S, Rand AG (2000) Evaluation of antibody immobilization methods for piezoelectric biosensor application. Biosens Bioelectron 15(11–12):615–621CrossRefGoogle Scholar
  15. 15.
    Danczyk R, Krieder B, North A, Webster T, HogenEsch H, Rundell A (2003) Comparison of antibody functionality using different immobilization methods. Biotechnol Bioeng 84(2):215–223CrossRefGoogle Scholar
  16. 16.
    Cheng C, Peng Y, Bai J, Zhang X, Liub Y, Fan X, Ning B, Gao Z (2014) Rapid detection of listeria monocytogenes in milk by self-assembled electrochemical immunosensor. Sensors Actuators B 190:900–906CrossRefGoogle Scholar
  17. 17.
    Luczak T, Osinska M (2017) New self-assembled layers composed with gold nanoparticles, cysteamine and dihydrolipoic acid deposited on bare gold template for highly sensitive and selective simultaneous sensing of dopamine in the presence of interfering ascorbic and uric acids. J Solid State Electrochem 21(3):747–758CrossRefGoogle Scholar
  18. 18.
    Yang Z, Gonzalez-Cortes A, Jourquin G, Viré J-C, Kauffmann J-M, Delplancke J-L (1995) Analytical application of self-assembled monolayers on gold electrodes: critical importance of surface pretreatment. Biosens Bioelectron 10(9):789–795CrossRefGoogle Scholar
  19. 19.
    Sun X, Zhu Y, Wang XY (2011) Amperometric Immunosensor based on a protein a/deposited gold nanocrystals modified electrode for Carbofuran detection. Sensors 11(12):11679–11691CrossRefGoogle Scholar
  20. 20.
    Salmain M, Ghasemi M, Boujday S, Pradier CM (2012) Elaboration of a reusable immunosensor for the detection of staphylococcal enterotoxin a ( SEA) in milk with a quartz crystal microbalance. Sensors Actuators B Chem 173:148–156CrossRefGoogle Scholar
  21. 21.
    Derkus B, Emregul K, Mazi H, Emregul E, Yumak T, Sinag A (2014) Protein a immunosensor for the detection of immunoglobulin G by impedance spectroscopy. Bioprocess Biosyst Eng 37(5):965–976CrossRefGoogle Scholar
  22. 22.
    Skottrup PD, Nicolaisen M, Justesen AF (2008) Towards on- site pathogen detection using antibody- based sensors. Biosens Bioelectron 24(3):339–348CrossRefGoogle Scholar
  23. 23.
    Green AA, Hughs WL (1955) Methods in enzymology, vol v. 1. Academic Press, New YorkGoogle Scholar
  24. 24.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  25. 25.
    Avrameas S (1969) Coupling of enzymes to proteins with glutaraldehyde: Use of the conjugates for the detection of antigens and antibodies. Immunochemistry 6:43–52CrossRefGoogle Scholar
  26. 26.
    Susmel S, Sullivan CK, Guilbault GG (2000) Human cytomegalovirus detection by a quartz crystal microbalance immunosensor. Enzym Microb Technol 27(9):639–645CrossRefGoogle Scholar
  27. 27.
    Tlili A, Abdelghani A, Hleli S, Maaref MA (2004) Electrical characterization of a thiol SAM on gold as a first step for the fabrication of immunosensors based on a quartz crystal microbalance. Sensors 4(6–7):105–114CrossRefGoogle Scholar
  28. 28.
    Dijksma M, Boukamp BA, Kamp B, van Bennekom WP (2002) Effect of hexacyanoferrate(ii/iii) on self-assembled monolayers of thioctic acid and 11-mercaptoundecanoic acid on gold. Langmuir 18:3105–3112CrossRefGoogle Scholar
  29. 29.
    Jung C, Dannenberger O, Xu Y, Buck M, Grunze M (1998) Self-assembled monolayers from organosulfur compounds: a comparison between sulfides, disulfides, and thiols. Langmuir 14:1103–1107CrossRefGoogle Scholar
  30. 30.
    Leopold MC, Bowden EF (2002) Influence of gold substrate topography on the voltammetry of cytochrome c adsorbed on carboxylic acid terminated self-assembled monolayers. Langmuir 18:2239–2245CrossRefGoogle Scholar
  31. 31.
    Mantzila AG, Maipa V, Prodromidis MI (2008) Development of a faradic impedimetric immunosensor for the detection of Salmonella typhimurium in milk. Anal Chem 80(4):1169CrossRefGoogle Scholar
  32. 32.
    Droz E, Taborelli M, Descouts P, Wells TNC, Werlen RC (1996) Covalent immobilization of immunoglobulins G and fab’ fragments on gold substates for scanning force microscopy imaging in liquids. J Vac Sci Technol B 14:1422–1426CrossRefGoogle Scholar
  33. 33.
    Björk I, Petersson B, Sjöquist J (1972) Some physicochemical properties of protein a from Staphylococcus Aureus. Eur J Biochem 29(3):579–584CrossRefGoogle Scholar
  34. 34.
    Gopinath SCB, Tang TH, Citartan M, Chen Y, Lakshmipriya T (2014) Current aspects in immunosensors. Biosens Bioelectron 57:292–302CrossRefGoogle Scholar
  35. 35.
    Furtado RF, Alves CR, Moreira ACO, Azevedo RM, Dutra RF (2012) A novel xyloglucan film-based biosensor for toxicity assessment of ricin in castor seed meal. Carbohydr Polym 89:586–591CrossRefGoogle Scholar
  36. 36.
    Barth A (2007) Infrared spectroscopy of proteins. Biochim Biophys Acta 1767:1073–1101CrossRefGoogle Scholar
  37. 37.
    Kong J, Yu S (2007) Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta Biochim Biophys Sin 39(8):549–559CrossRefGoogle Scholar
  38. 38.
    Mehrvar M, Abdi M (2004) Recent developments, characteristics, and potential applications of electrochemical biosensors. Anal Sci 20:1113–1126CrossRefGoogle Scholar
  39. 39.
    Rosatto SS, Freire RS, Durán N, Kubota LT (2001) Amperometric biosensors for phenolic compounds determination in the environmental interess samples. Química Nova 24:77–86CrossRefGoogle Scholar
  40. 40.
    Lei C-X, Hu S-Q, Gao N, Shen G-L, Yu R-Q (2004) An amperometric hydrogen peroxide biosensor based on immobilizing horseradish peroxidase to a nano-au monolayer supported by sol–gel derived carbon ceramic electrode. Bioelectrochemistry 65:33–39CrossRefGoogle Scholar
  41. 41.
    Oh B, Kim Y, Park K, Lee W, Choi JW (2004) Surface plasmon resonance immunosensor for the detection of Salmonella typhimurium. Biosens Bioelectron 19(11):1497–1504CrossRefGoogle Scholar
  42. 42.
    Oh BK, Lee W, Lee WH, Choi JW, Kim YK (2004) Surface plasmon resonance immunosensor using self-assembled protein G for the detection of Salmonella paratyphi. J Biotechnol 111:1–8CrossRefGoogle Scholar
  43. 43.
    Bae Y, Park K, Oh B, Lee W, Choi JW (2005) Immunosensor for detection of Salmonella typhimurium based on imaging ellipsometry. Colloids Surf A Physicochem Eng Asp 257:19–23CrossRefGoogle Scholar
  44. 44.
    O’Shannessy DJ, Brigham-Burke M, Peck K (1992) Immobilization chemistries suitable for use in the BlAcore surface Plasmon resonance detector. Anal Biochem 205:132–136CrossRefGoogle Scholar
  45. 45.
    Barie N, Rapp M (2001) Covalent bound sensing layers on surface acoustic wave (SAW) biosensors. Biosens Bioelectron 16:979–987CrossRefGoogle Scholar
  46. 46.
    Lee KM, Runyon M, Herrman TJ, Hsieh J, Phillips R (2015) Review of Salmonella detection and identification methods: aspects of rapid emergency response and food safety. Food Control 47:264–276CrossRefGoogle Scholar
  47. 47.
    Knirel YA, Kocharova NA, Bystrova OV, Katzenellenbogen E, Gamian A (2002) Structures and serology of the O-specific polysaccharides of bacteria of the genus Citrobacter. Arch Immunol Ther Exp 50(6):379–391Google Scholar
  48. 48.
    Péterfi Z, Kustos I, Kilár F, Kocsis B (2007) Microfluidic chip analysis of outer membrane proteins responsible for serological cross-reaction between three gram-negative bacteria: Proteus Morganii O34, Escherichia Coli O111 and Salmonella Adelaide O35. J Chromatogr A 1155(1):214–217CrossRefGoogle Scholar
  49. 49.
    Kim YS, Raston NHA, Gu MB (2016) Aptamer-based nanobiosensors. Biosens Bioeletron 76:2–19CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • A. M. A. Melo
    • 1
  • D. L. Alexandre
    • 2
  • M. R. F. Oliveira
    • 2
  • R. F. Furtado
    • 3
  • M. F. Borges
    • 3
  • P. R. V. Ribeiro
    • 3
  • A. Biswas
    • 4
  • H. N. Cheng
    • 5
  • C. R. Alves
    • 2
  • E. A. T. Figueiredo
    • 1
  1. 1.Department of Science and Food TechnologyFederal University of CearáFortalezaBrazil
  2. 2.Department of ChemistryFortalezaBrazil
  3. 3.Embrapa Tropical AgroindustryFortalezaBrazil
  4. 4.USDA Agricultural Research ServiceNational Center for Agricultural Utilization ResearchPeoriaUSA
  5. 5.USDA Agricultural Research Service, Southern Regional Research CenterNew OrleansUSA

Personalised recommendations