An ELISA Lab-on-a-Chip (ELISA-LOC)

  • Avraham RasoolyEmail author
  • Hugh A. Bruck
  • Yordan Kostov
Part of the Methods in Molecular Biology book series (MIMB, volume 949)


Laminated object manufacturing (LOM) technology using polymer sheets is an easy and affordable method for rapid prototyping of Lab-on-a-Chip (LOC) systems. It has recently been used to fabricate a miniature 96 sample ELISA lab-on-a-chip (ELISA-LOC) by integrating the washing step directly into an ELISA plate. LOM has been shown to be capable of creating complex 3D microfluidics through the assembly of a stack of polymer sheets with features generated by laser micromachining and by bonding the sheets together with adhesive. A six layer ELISA-LOC was fabricated with an acrylic (poly(methyl methacrylate) (PMMA)) core and five polycarbonate layers micromachined by a CO2 laser with simple microfluidic features including a miniature 96-well sample plate. Immunological assays can be carried out in several configurations (1 × 96 wells, 2 × 48 wells, or 4 × 24 wells). The system includes three main functional elements: (1) a reagent loading fluidics module, (2) an assay and detection wells plate, and (3) a reagent removal fluidics module. The ELISA-LOC system combines several biosensing elements: (1) carbon nanotube (CNT) technology to enhance primary antibody immobilization, (2) sensitive ECL (electrochemiluminescence) detection, and (3) a charge-coupled device (CCD) detector for measuring the light signal generated by ECL. Using a sandwich ELISA assay, the system detected Staphylococcal enterotoxin B (SEB) at concentrations as low as 0.1 ng/ml, a detection level similar to that reported for conventional ELISA. ELISA-LOC can be operated by a syringe and does not require power for operation. This simple point-of-care (POC) system is useful for carrying out various immunological assays and other complex medical assays without the laboratory required for conventional ELISA, and therefore may be more useful for global healthcare delivery.

Key words

ELISA Lamination Charge-coupled device Micromachining Microfluidics Staphylococcal enterotoxins Enhanced chemiluminescence Carbon nanotubes Point-of-care-settings Food safety 


  1. 1.
    Engvall E, Perlmann P (1971) Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G. Immunochemistry 8:871–874CrossRefGoogle Scholar
  2. 2.
    Van Weemen BK, Schuurs AH (1971) Immunoassay using antigen-enzyme conjugates. FEBS Lett 15:232–236CrossRefGoogle Scholar
  3. 3.
    Ihara M, Yoshikawa A, Wu Y, Takahashi H, Mawatari K, Shimura K, Sato K, Kitamori T, Ueda H (2010) Micro OS-ELISA: rapid noncompetitive detection of a small biomarker peptide by open-sandwich enzyme-linked immunosorbent assay (OS-ELISA) integrated into microfluidic device. Lab Chip 10:92–100CrossRefGoogle Scholar
  4. 4.
    Gao Y, Sherman PM, Sun Y, Li D (2008) Multiplexed high-throughput electrokinetically-controlled immunoassay for the detection of specific bacterial antibodies in human serum. Anal Chim Acta 606:98–107CrossRefGoogle Scholar
  5. 5.
    Kong J, Jiang L, Su X, Qin J, Du Y, Lin B (2009) Integrated microfluidic immunoassay for the rapid determination of clenbuterol. Lab Chip 9:1541–1547CrossRefGoogle Scholar
  6. 6.
    Tseng YT, Yang CS, Tseng FG (2009) A perfusion-based micro opto-fluidic system (PMOFS) for continuously in-situ immune sensing. Lab Chip 9:2673–2682CrossRefGoogle Scholar
  7. 7.
    Lee BS, Lee JN, Park JM, Lee JG, Kim S, Cho YK, Ko C (2009) A fully automated immunoassay from whole blood on a disc. Lab Chip 9:1548–1555CrossRefGoogle Scholar
  8. 8.
    Javanmard M, Talasaz AH, Nemat-Gorgani M, Pease F, Ronaghi M, Davis RW (2009) Electrical detection of protein biomarkers using bioactivated microfluidic channels. Lab Chip 9:1429–1434CrossRefGoogle Scholar
  9. 9.
    Tachi T, Kaji N, Tokeshi M, Baba Y (2009) Microchip-based homogeneous immunoassay using fluorescence polarization spectroscopy. Lab Chip 9:966–971CrossRefGoogle Scholar
  10. 10.
    Liu C, Qiu X, Ongagna S, Chen D, Chen Z, Abrams WR, Malamud D, Corstjens PL, Bau HH (2009) A timer-actuated immunoassay cassette for detecting molecular markers in oral fluids. Lab Chip 9:768–776CrossRefGoogle Scholar
  11. 11.
    Meagher RJ, Hatch AV, Renzi RF, Singh AK (2008) An integrated microfluidic platform for sensitive and rapid detection of biological toxins. Lab Chip 8:2046–2053CrossRefGoogle Scholar
  12. 12.
    Reichmuth DS, Wang SK, Barrett LM, Throckmorton DJ, Einfeld W, Singh AK (2008) Rapid microchip-based electrophoretic immunoassays for the detection of swine influenza virus. Lab Chip 8:1319–1324CrossRefGoogle Scholar
  13. 13.
    Luo Y, Yu F, Zare RN (2008) Microfluidic device for immunoassays based on surface plasmon resonance imaging. Lab Chip 8:694–700CrossRefGoogle Scholar
  14. 14.
    Choi Y, Kang T, Lee LP (2009) Plasmon resonance energy transfer (PRET)-based molecular imaging of cytochrome c in living cells. Nano Lett 9:85–90CrossRefGoogle Scholar
  15. 15.
    Lucas LJ, Chesler JN, Yoon JY (2007) Lab-on-a-chip immunoassay for multiple antibodies using microsphere light scattering and quantum dot emission. Biosens Bioelectron 23:675–681CrossRefGoogle Scholar
  16. 16.
    Stevens DY, Petri CR, Osborn JL, Spicar-Mihalic P, McKenzie KG, Yager P (2008) Enabling a microfluidic immunoassay for the developing world by integration of on-card dry reagent storage. Lab Chip 8:2038–2045CrossRefGoogle Scholar
  17. 17.
    Tang D, Tang J, Su B, Ren J, Chen G (2009) Simultaneous determination of five-type hepatitis virus antigens in 5 min using an integrated automatic electrochemical immunosensor array. Biosens Bioelectron 25(7):1658–1662CrossRefGoogle Scholar
  18. 18.
    Mujika M, Arana S, Castano E, Tijero M, Vilares R, Ruano-Lopez JM, Cruz A, Sainz L, Berganza J (2009) Magnetoresistive immunosensor for the detection of Escherichia coli O157:H7 including a microfluidic network. Biosens Bioelectron 24:1253–1258CrossRefGoogle Scholar
  19. 19.
    Lee SM, Hwang KS, Yoon HJ, Yoon DS, Kim SK, Lee YS, Kim TS (2009) Sensitivity enhancement of a dynamic mode microcantilever by stress inducer and mass inducer to detect PSA at low picogram levels. Lab Chip 9:2683–2690CrossRefGoogle Scholar
  20. 20.
    Sapsford KE, Francis J, Sun S, Kostov Y, Rasooly A (2009) Miniaturized 96-well ELISA chips for staphylococcal enterotoxin B detection using portable colorimetric detector. Anal Bioanal Chem 394:499–505CrossRefGoogle Scholar
  21. 21.
    Yang M, Kostov Y, Bruck HA, Rasooly A (2008) Carbon nanotubes with enhanced chemiluminescence immunoassay for CCD-based detection of Staphylococcal enterotoxin B in food. Anal Chem 80:8532–8537CrossRefGoogle Scholar
  22. 22.
    Sapsford KE, Sun S, Francis J, Sharma S, Kostov Y, Rasooly A (2008) A fluorescence detection platform using spatial electroluminescent excitation for measuring botulinum neurotoxin A activity. Biosens Bioelectron 24:618–625CrossRefGoogle Scholar
  23. 23.
    Yang M, Kostov Y, Bruck HA, Rasooly A (2009) Gold nanoparticle-based enhanced chemiluminescence immunosensor for detection of Staphylococcal Enterotoxin B (SEB) in food. Int J Food Microbiol 133:265–271CrossRefGoogle Scholar
  24. 24.
    Sun S, Ossandon M, Kostov Y, Rasooly A (2009) Lab-on-a-chip for botulinum neurotoxin a (BoNT-A) activity analysis. Lab Chip 9:3275–3281CrossRefGoogle Scholar
  25. 25.
    Sun S, Yang M, Kostov Y, Rasooly A (2010) ELISA-LOC: lab-on-a-chip for enzyme-linked immunodetection. Lab Chip 10:2093–2100CrossRefGoogle Scholar
  26. 26.
    Xia Y, Kim E, Zhao XM, Rogers JA, Prentiss M, Whitesides GM (1996) Complex optical surfaces formed by replica molding against elastomeric masters. Science 273:347–349CrossRefGoogle Scholar
  27. 27.
    Delamarche E, Bernard A, Schmid H, Michel B, Biebuyck H (1997) Patterned delivery of immunoglobulins to surfaces using microfluidic networks. Science 276:779–781CrossRefGoogle Scholar
  28. 28.
    Irawan R, Tjin SC, Yager P, Zhang D (2005) Cross-talk problem on a fluorescence multi-channel microfluidic chip system. Biomed Microdevices 7:205–211CrossRefGoogle Scholar
  29. 29.
    Schilling EA, Kamholz AE, Yager P (2002) Cell lysis and protein extraction in a microfluidic device with detection by a fluorogenic enzyme assay. Anal Chem 74:1798–1804CrossRefGoogle Scholar
  30. 30.
    Munson MS, Hasenbank MS, Fu E, Yager P (2004) Suppression of non-specific adsorption using sheath flow. Lab Chip 4:438–445CrossRefGoogle Scholar
  31. 31.
    Rossier JS, Schwarz A, Reymond F, Ferrigno R, Bianchi F, Girault HH (1999) Microchannel networks for electrophoretic separations. Electrophoresis 20:727–731CrossRefGoogle Scholar
  32. 32.
    Rossier J, Reymond F, Michel PE (2002) Polymer microfluidic chips for electrochemical and biochemical analyses. Electrophoresis 23:858–867CrossRefGoogle Scholar
  33. 33.
    Taitt CR, Anderson GP, Ligler FS (2005) Evanescent wave fluorescence biosensors. Biosens Bioelectron 20:2470–2487CrossRefGoogle Scholar
  34. 34.
    Ngundi MM, Qadri SA, Wallace EV, Moore MH, Lassman ME, Shriver-Lake LC, Ligler FS, Taitt CR (2006) Detection of deoxynivalenol in foods and indoor air using an array biosensor. Environ Sci Technol 40:2352–2356CrossRefGoogle Scholar
  35. 35.
    Moreno-Bondi MC, Taitt CR, Shriver-Lake LC, Ligler FS (2006) Multiplexed measurement of serum antibodies using an array biosensor. Biosens Bioelectron 21:1880–1886CrossRefGoogle Scholar
  36. 36.
    Ligler FS, Sapsford KE, Golden JP, Shriver-Lake LC, Taitt CR, Dyer MA, Barone S, Myatt CJ (2007) The array biosensor: portable, automated systems. Anal Sci 23:5–10CrossRefGoogle Scholar
  37. 37.
    Yang M, Kostov Y, Rasooly A (2008) Carbon nanotubes based optical immunodetection of Staphylococcal Enterotoxin B (SEB) in food. Int J Food Microbiol 127:78–83CrossRefGoogle Scholar
  38. 38.
    Hu D, Han H, Zhou R, Dong F, Bei W, Jia F, Chen H (2008) Gold(III) enhanced chemiluminescence immunoassay for detection of antibody against ApxIV of Actinobacillus pleuropneumoniae. Analyst 133:768–773CrossRefGoogle Scholar
  39. 39.
    Rubtsova M, Kovba GV, Egorov AM (1998) Chemiluminescent biosensors based on porous supports with immobilized peroxidase. Biosens Bioelectron 13:75–85CrossRefGoogle Scholar
  40. 40.
    Archer DL, Young FE (1988) Contemporary issues: diseases with a food vector. Clin Microbiol Rev 1:377–398Google Scholar
  41. 41.
    Olsen SJ, MacKinnon LC, Goulding JS, Bean NH, Slutsker L (2000) Surveillance for foodborne-disease outbreaks—United States, 1993–1997. MMWR CDC Surveill Summ 49:1–62Google Scholar
  42. 42.
    Bean NH, Goulding JS, Lao C, Angulo FJ (1996) Surveillance for foodborne-disease outbreaks—United States, 1988–1992. MMWR CDC Surveill Summ 45:1–66Google Scholar
  43. 43.
    Bunning VK, Lindsay JA, Archer DL (1997) Chronic health effects of microbial foodborne disease. World Health Stat Q 50:51–56Google Scholar
  44. 44.
    Garthright WE, Archer DL, Kvenberg JE (1988) Estimates of incidence and costs of intestinal infectious diseases in the United States. Public Health Rep 103:107–115Google Scholar
  45. 45.
    Asao T, Kumeda Y, Kawai T, Shibata T, Oda H, Haruki K, Nakazawa H, Kozaki S (2003) An extensive outbreak of staphylococcal food poisoning due to low-fat milk in Japan: estimation of enterotoxin A in the incriminated milk and powdered skim milk. Epidemiol Infect 130:33–40CrossRefGoogle Scholar
  46. 46.
    Breuer K, Wittmann M, Bosche B, Kapp A, Werfel T (2000) Severe atopic dermatitis is associated with sensitization to staphylococcal enterotoxin B (SEB). Allergy 55:551–555CrossRefGoogle Scholar
  47. 47.
    Bunikowski R, Mielke M, Skarabis H, Herz U, Bergmann RL, Wahn U, Renz H (1999) Prevalence and role of serum IgE antibodies to the Staphylococcus aureus-derived superantigens SEA and SEB in children with atopic dermatitis. J Allergy Clin Immunol 103:119–124CrossRefGoogle Scholar
  48. 48.
    Mempel M, Lina G, Hojka M, Schnopp C, Seidl HP, Schafer T, Ring J, Vandenesch F, Abeck D (2003) High prevalence of superantigens associated with the egc locus in Staphylococcus aureus isolates from patients with atopic eczema. Eur J Clin Microbiol Infect Dis 22:306–309Google Scholar
  49. 49.
    Howell MD, Diveley JP, Lundeen KA, Esty A, Winters ST, Carlo DJ, Brostoff SW (1991) Limited T-cell receptor beta-chain heterogeneity among interleukin 2 receptor-positive synovial T cells suggests a role for superantigen in rheumatoid arthritis. Proc Natl Acad Sci U S A 88:10921–10925CrossRefGoogle Scholar
  50. 50.
    Uematsu Y, Wege H, Straus A, Ott M, Bannwarth W, Lanchbury J, Panayi G, Steinmetz M (1991) The T-cell-receptor repertoire in the synovial fluid of a patient with rheumatoid arthritis is polyclonal. Proc Natl Acad Sci U S A 88:8534–8538CrossRefGoogle Scholar
  51. 51.
    Herz U, Bunikowski R, Mielke M, Renz H (1999) Contribution of bacterial superantigens to atopic dermatitis. Int Arch Allergy Immunol 118:240–241CrossRefGoogle Scholar
  52. 52.
    Wiener SL (1996) Strategies for the prevention of a successful biological warfare aerosol attack. Mil Med 161:251–256Google Scholar
  53. 53.
    Ler SG, Lee FK, Gopalakrishnakone P (2006) Trends in detection of warfare agents. Detection methods for ricin, staphylococcal enterotoxin B and T-2 toxin. J Chromatogr A 1133:1–12CrossRefGoogle Scholar
  54. 54.
    Henghold WB II (2004) Other biologic toxin bioweapons: ricin, staphylococcal enterotoxin B, and trichothecene mycotoxins. Dermatol Clin 22:257–262, vCrossRefGoogle Scholar
  55. 55.
    Rosenbloom M, Leikin JB, Vogel SN, Chaudry ZA (2002) Biological and chemical agents: a brief synopsis. Am J Ther 9:5–14CrossRefGoogle Scholar
  56. 56.
    Sergeev N, Volokhov D, Chizhikov V, Rasooly A (2004) Simultaneous analysis of multiple staphylococcal enterotoxin genes by an oligonucleotide microarray assay. J Clin Microbiol 42:2134–2143CrossRefGoogle Scholar
  57. 57.
    Jarraud S, Peyrat MA, Lim A, Tristan A, Bes M, Mougel C, Etienne J, Vandenesch F, Bonneville M, Lina G (2001) egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus. J Immunol 166:669–677Google Scholar
  58. 58.
    Jarraud S, Mougel C, Thioulouse J, Lina G, Meugnier H, Forey F, Nesme X, Etienne J, Vandenesch F (2002) Relationships between Staphylococcus aureus genetic background, virulence factors, agr groups (alleles), and human disease. Infect Immun 70:631–641CrossRefGoogle Scholar
  59. 59.
    Omoe K, Ishikawa M, Shimoda Y, Hu DL, Ueda S, Shinagawa K (2002) Detection of seg, seh, and sei genes in Staphylococcus aureus isolates and determination of the enterotoxin productivities of S. aureus isolates Harboring seg, seh, or sei genes. J Clin Microbiol 40:857–862CrossRefGoogle Scholar
  60. 60.
    Liu G, Lin Y (2006) Biosensor based on self-assembling acetylcholinesterase on carbon nanotubes for flow injection/amperometric detection of organophosphate pesticides and nerve agents. Anal Chem 78:835–843CrossRefGoogle Scholar
  61. 61.
    Bennett RW (2005) Staphylococcal enterotoxin and its rapid identification in foods by enzyme-linked immunosorbent assay-based methodology. J Food Prot 68:1264–1270Google Scholar
  62. 62.
    Miyamoto T, Kamikado H, Kobayashi H, Honjoh K, Iio M (2003) Immunomagnetic flow cytometric detection of staphylococcal enterotoxin B in raw and dry milk. J Food Prot 66:1222–1226Google Scholar
  63. 63.
    Pan TM, Yu YL, Chiu SI, Lin SS (1996) [Comparison of immunoassay kits for detection of staphylococcal enterotoxins produced by Staphylococcus aureus]. Zhonghua Minguo wei sheng wu ji mian yi xue za zhi (Chinese journal of microbiology and immunology) 29:100–107Google Scholar
  64. 64.
    Park CE, Akhtar M, Rayman MK (1994) Evaluation of a commercial enzyme immunoassay kit (RIDASCREEN) for detection of staphylococcal enterotoxins A, B, C, D, and E in foods. Appl Environ Microbiol 60:677–681Google Scholar
  65. 65.
    Park CE, Warburton D, Laffey PJ (1996) A collaborative study on the detection of staphylococcal enterotoxins in foods by an enzyme immunoassay kit (RIDASCREEN). Int J Food Microbiol 29:281–295CrossRefGoogle Scholar
  66. 66.
    Vernozy-Rozand C, Mazuy-Cruchaudet C, Bavai C, Richard Y (2004) Comparison of three immunological methods for detecting staphylococcal enterotoxins from food. Lett Appl Microbiol 39:490–494CrossRefGoogle Scholar
  67. 67.
    Wieneke AA (1991) Comparison of four kits for the detection of staphylococcal enterotoxin in foods from outbreaks of food poisoning. Int J Food Microbiol 14:305–312CrossRefGoogle Scholar
  68. 68.
    Hawkins KR, Yager P (2003) Nonlinear decrease of background fluorescence in polymer thin-films—a survey of materials and how they can complicate fluorescence detection in microTAS. Lab Chip 3:248–252CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media,LLC 2013

Authors and Affiliations

  • Avraham Rasooly
    • 1
    • 2
    Email author
  • Hugh A. Bruck
    • 3
  • Yordan Kostov
    • 4
    • 5
  1. 1.Division of Biology, Office of Science and EngineeringFDA Center for Devices and Radiological Health (CDRH)Silver SpringUSA
  2. 2.National Cancer InstituteRockvilleUSA
  3. 3.Department of Mechanical EngineeringUniversity of MarylandCollege ParkUSA
  4. 4.Steven Sun Division of Biology Office of Science and EngineeringFDA Center for Devices and Radiological Health (CDRH)Silver SpringUSA
  5. 5.University of Maryland Baltimore CountyBaltimore CountyUSA

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