Environmental Science and Pollution Research

, Volume 24, Issue 1, pp 15–24 | Cite as

Sorption of his-tagged Protein G and Protein G onto chitosan/divalent metal ion sorbent used for detection of microcystin-LR

  • Hary Demey
  • Scherrine A. Tria
  • Romain Soleri
  • Anthony Guiseppi-Elie
  • Ingrid Bazin
In-line Multiplexed Biosensing


A highly sensitive, specific, simple, and rapid chemiluminescence enzyme immunoassay (CLEIA) was developed for the determination of microcystin-LR (MC-LR) by using strategies for oriented immobilization of functionally intact polyclonal antibodies on chitosan surface. Several physicochemical parameters such as metal ion adsorption, hexahistidine-tagged Protein G sorption, the dilution ratio polyclonal antibody concentration, and peroxidase-labeled MC-LR concentration were studied and optimized. The sorption in batch system of G-histidine and G-proteins was studied on a novel sorbent consisting of chitosan/divalent metal ions. Transition metals as Ni++ and Zn++ were immobilized through interaction with –NH2 groups of chitosan in order to supply a material capable to efficiently remove the proteins from aqueous solutions. The maximum uptake of divalent metals onto the chitosan material was found to be 230 mg g−1 for Zn++ and 62 mg g−1 for Ni++. Experimental data were evaluated using the Langmuir and Freundlich models; the results were well fitted with the Langmuir model; chitosan/Ni++ foam was found to be the best sorbent for G-protein, maximum sorption capacity obtained was 17 mg g−1, and chitosan/Zn++ was found to be the best for G-histidine with a maximum sorption capacity of 44 mg g−1. Kinetic data was evaluated with pseudo-first- and pseudo-second-order models; the sorption kinetics were in all cases better represented by a pseudo-second-order model. Under optimum conditions, the calibration curve obtained for MC-LR gave detection limits of 0.5 ± 0.06 μg L−1, the 50 % inhibition concentration (IC50) was 2.75 ± 0.03 μg L−1, and the quantitative detection range was 0.5–25 μg L−1. The limit of detection (LOD) attained from the calibration curves and the results obtained demonstrate the potential use of CLEIA with chitosan support as a screening tool for the analysis of pollutants in environmental samples.


Chitosan Microcystin-LR Antibody orientation Protein G Affinity tag 



AGE acknowledges receipt of a visiting professorship from EMA. This work was financially supported by the French National Research Agency via funding for the COMBITOX project (ANR-11-ECOT-009-04)


  1. Agarwal M, Das A, Singh AS (2010). High‐dose hook effect in prolactin macroadenomas: a diagnostic concern. J Hum Reprod Sci 3:160–161Google Scholar
  2. Ahmed SR, Kelly AB, Barbari TA (2006) Controlling the orientation of immobilized proteins on an affinity membrane through chelation of a histidine tag to a chitosan-Ni++ surface. J Membr Sci 280:553–559CrossRefGoogle Scholar
  3. Campàs M, Marty J (2007) Highly sensitive amperometric immunosensors for microcystin detection in algae. Biosens Bioelectron 22:1034–1040CrossRefGoogle Scholar
  4. Carmichael WW, Azevedo SM, An JS, Molica RJ, Jochimsen EM, Lau S, Rinehart KL, Shaw GR, Eaglesham GK (2001) Human fatalities from cyanobacteria: chemical and biological evidence for cyanotoxins. Environ Health Perspect 109:663–668CrossRefGoogle Scholar
  5. Catanante G, Espin L, Marty JL (2015) Sensitive biosensor based on recombinant PP1α for microcystin detection. Biosens Bioelectron 67:700–707CrossRefGoogle Scholar
  6. Delehanty JB, Ligler FS (2002) A microarray immunoassay for simultaneous detection of proteins and bacteria. Anal Chem 74:5681–5687CrossRefGoogle Scholar
  7. Demey H, Vincent T, Ruiz M, Sastre AM, Guibal E (2014) Development of a new chitosan/Ni(OH)2-based sorbent for boron removal. Chem Eng J 244:576–586CrossRefGoogle Scholar
  8. Eser A, Tirtom N, Aydemir T, Becerik S, Dinçer A (2012) Removal of nickel(II) ions by histidine modified chitosan beads. Chem Eng J 210:590–596CrossRefGoogle Scholar
  9. Freundlich HMF (1906) Über die adsorption in lösungen. Z Phys Chem 57A:385–470Google Scholar
  10. Gehringer MM, Kewada V, Coates N, Downing TG (2003) The use of Lepidium sativum in a plant bioassay system for the detection of microcystin-LR. Toxicon 41:871–876CrossRefGoogle Scholar
  11. Guibal E, Larkin A, Vincent T, Tobin JM (1999) Chitosan sorbents for platinum sorption from dilute solutions. Ind Eng Chem Res 38:401–412CrossRefGoogle Scholar
  12. Ho YS, McKay G (1998) Sorption of dye from aqueous solution by peat. Chem Eng J 70:115–124CrossRefGoogle Scholar
  13. Ingavle GC, Baillie LW, Zheng Y, Lis EK, Savina IN, Howell CA, Mikhalovsky SV, Sandeman SR (2015) Affinity binding of antibodies to supermacroporous cryogel adsorbents with immobilized protein A for removal of anthrax toxin protective antigen. Biomaterials 50:140–153CrossRefGoogle Scholar
  14. Johnson CP, Jensen IE, Prakasam A, Vijayendran R, Leckband D (2003) Engineered protein A for the orientational control of immobilized proteins. Bioconjugate Chem 14:974–978CrossRefGoogle Scholar
  15. Juang RS, Shao HJ (2002) Effect of pH on competitive adsorption of Cu(II), Ni(II), and Zn(II) from water onto chitosan beads. Adsorption 8:71–78CrossRefGoogle Scholar
  16. Knezevic V, Leethanakul C, Bichsel VE, Worth JM, Prabhu VV, Gutkind JS, Liotta LA, Munson PJ, Petricoin EF, Krizman DB (2001) Proteomic profiling of the cancer microenvironment by antibody arrays. Proteomics 1:1271–1278CrossRefGoogle Scholar
  17. Lagergreen S (1898) Zur theorie der sogenannten adsorption gelöster stoffe. Kungliga Svenska Vetenskapsakademiens. Handl 24:1–39Google Scholar
  18. Langmuir I (1918) The adsorption of gases on plane surfaces of glass, mica and platinum. J Am Chem Soc 40:1361–1403CrossRefGoogle Scholar
  19. Lawrence JF, Niedzwiadek B, Menard C, Lau BP, Lewis D, Kuper-Goodman T, Carbone S, Holmes C (2001) Comparison of liquid chromatography/mass spectrometry, ELISA, and phosphatase assay for the determination of microcystins in blue‐green algae products. J AOAC Int 10:1035–1044Google Scholar
  20. Liu M, Zhao H, Chen S, Yu H, Quan X (2012) Colloidal graphene as a transducer in homogeneous fluorescence-based immunosensor for rapid and sensitive analysis of microcystin-LR. Environ Sci Technol 46:12567–12574CrossRefGoogle Scholar
  21. Makaraviciute A, Ramanaviciene A (2013) Site-directed antibody immobilization techniques for immunosensors. Biosens Bioelectron 50:460–471CrossRefGoogle Scholar
  22. McElhiney J, Lawton LA (2003) Detection of the cyanobacterial hepatotoxins microcystins. Toxicol Appl Pharmacol 203:219–230CrossRefGoogle Scholar
  23. Murphy C, Stack E, Krivelo S, McPartlin DA, Byrne B, Greef C, Lochhead MJ, Husar G, Devlin S, Elliott CT, O’Kennedy RJ (2015) Detection of the cyanobacterial toxin, microcystin-LR, using a novel recombinant antibody-based optical-planar waveguide platform. Biosens Bioelectron 67:708–714CrossRefGoogle Scholar
  24. Naik KBK, Raju S, Kumar BA, Rao GN (2011) Speciation studies of l-histidine complexes of Pb(II), Cd(II) and Hg(II) in Dioxan-water mixtures. Der Pharma Chem 3:194–205Google Scholar
  25. Namasivayam C, Yamuna RT (1995) Adsorption of direct red 12 B by biogas residual slurry: equilibrium and rate processes. Environ Pollut 89:1–7CrossRefGoogle Scholar
  26. Nielsen UB, Geierstanger BH (2004) Multiplexed sandwich assays in microarray format. J Immunol Meth 290:107–120CrossRefGoogle Scholar
  27. Özturk N, Kavak D (2005) Adsorption of boron from aqueous solutions using fly ash: batch and column studies. J Hazard Mater B 127:81–88Google Scholar
  28. Peluso P, Wilson DS, Do D, Tran H, Venkatasubbaiah M, Quincy D, Heidecker B, Poindexter K, Tolani N, Phelan M, Witte K, Jung LS, Wagner P, Nock S (2003) Optimizing antibody immobilization strategies for the construction of protein microarrays. Anal Biochem 312:113–124CrossRefGoogle Scholar
  29. Plaza-Cazon J, Bernadelli C, Viera M, Donati E, Guibal E (2012) Zinc and cadmium biosorption by untreated and calcium-treated Macrocystis pyrifera in a batch system. Bioresour Technol 116:195–203CrossRefGoogle Scholar
  30. Ruiz M, Sastre AM, Zikan MC, Guibal E (2001) Palladium sorption on glutaraldehyde-crosslinked chitosan in fixed-bed systems. J Appl Polym Sci 81:153–165CrossRefGoogle Scholar
  31. Shirvani M, Sherkat Z, Khalili B, Bakhtiary S (2015) Sorption of Pb(II) on polygorskite and sepiolite in the presence of amino acids: equilibria and kinetics. Geoderma 249–250:21–27CrossRefGoogle Scholar
  32. Sieroslawska A (2014) Evaluation of usefulness of Microbial Assay for Risk Assessment (MARA) in the cyanobacterial toxicity estimation. Environ Monit Assess 186:4629–4636CrossRefGoogle Scholar
  33. Soleri R, Demey H, Tria SA, Guiseppi-Elie A, Hassine AI, Gonzalez C, Bazin I (2015) Peptide conjugated chitosan foam as a novel approach for capture-purification and rapid detection of hapten—example of ochratoxin A. Biosens Bioelectron 67:634–641CrossRefGoogle Scholar
  34. Spoof L, Neffling MR, Meriluoto J (2010) Fast separation of microcystins and nodularins on narrow-bore reversed-phase columns coupled to a conventional HPLC system. Toxicon 55:954–964CrossRefGoogle Scholar
  35. Tsutsumi E, Henares TG, Funano S, Kawamura K, Endo T, Hisamoto H (2012) Single-step sandwich immunoreaction in a square glass capillary immobilizing capture and enzyme-linked antibodies for simplified enzyme-linked immunosorbent assay. Anal Sci 28:51–56CrossRefGoogle Scholar
  36. Wacker R, Schroder H, Niemeyer CM (2004) Performance of antibody microarrays fabricated by either DNA-directed immobilization, direct spotting, or streptavidin-biotin attachment: a comparative study. Anal Biochem 330:281–287CrossRefGoogle Scholar
  37. Wan MW, Kan CC, Rogel BD, Dalida MLP (2010) Adsorption of copper (II) and lead (II) ions from aqueous solution on chitosan-coated sand. Carbohydr Polym 80:891–899CrossRefGoogle Scholar
  38. WHO (2004) Guidelines for drinking water quality, 3rd edn. World Health Organization, GenevaGoogle Scholar
  39. Wold ED, McBride R, Axup JY, Kazane SA, Smider VV (2015) Antibody microarrays utilizing site-specific antibody-oligonucleotide conjugates. Bioconjug Chem 26:807–811CrossRefGoogle Scholar
  40. Wu JY, Xu QJ, Gao G, Shen JH (2006) Evaluating genotoxicity associated with microcystin-LR and its risk to source water safety in Meiliang Bay, Taihu Lake. Environ Toxicol 21:250–255CrossRefGoogle Scholar
  41. Yang HM, Liang SJ, Tang JB, Chen Y, Cheng YZ (2013) Immobilization of unraveled immunoglobulin G using well-oriented ZZ-His protein on functionalized microtiter plate for sensitive immunoassay. Anal Biochem 432:134–138CrossRefGoogle Scholar
  42. Zeck A, Weller MG, Niessner R (2001) Multidimensional biochemical detection of microcystins in liquid chromatography. Anal Chem 73:5509–5517CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Hary Demey
    • 1
  • Scherrine A. Tria
    • 2
  • Romain Soleri
    • 2
  • Anthony Guiseppi-Elie
    • 3
  • Ingrid Bazin
    • 2
  1. 1.École des Mines d’Alès, Centre des Matériaux des Mines d’AlèsAlès CEDEXFrance
  2. 2.École des Mines d’Alès, Laboratoire de Génie de L’Environnement IndustrielAlès CEDEXFrance
  3. 3.Department of Biomedical Engineering, The Dwight Look College of EngineeringTexas A&M UniversityCollege StationUSA

Personalised recommendations