Multiplex Immunoassays

  • Sandeep Kumar Vashist


Multiplex immunoassays (IAs) refer to IA formats that can simultaneously determine many analytes in a single sample. They are becoming critically important in healthcare for the diagnosis of complex diseases, which require the simultaneous monitoring of multiple disease biomarkers. The ongoing research efforts are based on the determination of clinical scores for such complex diseases by assigning appropriate weightages to various biomarkers based on their contribution to the disease. Although a wide range of multiplex IA formats have been demonstrated by researchers with few of them commercialized successfully, there is still a need for the development of advanced and bioanalytically superior multiplex IA formats that are clinically and commercially viable. Further, the multiplex IAs should align well with the established and clinically accredited IAs. We provide here an overview of various multiplex IA formats and technologies together with the challenges involved, prospects, and guided insights.


Multiplex detection Immunoassays Bioanalytical platforms Assay concepts Clinical score 


  1. 1.
    Jung W, Han J, Choi J-W, Ahn CH. Point-of-care testing (POCT) diagnostic systems using microfluidic lab-on-a-chip technologies. Microelectron Eng. 2015;132:46–57.CrossRefGoogle Scholar
  2. 2.
    Spindel S, Sapsford K. Evaluation of optical detection platforms for multiplexed detection of proteins and the need for point-of-care biosensors for clinical use. Sensors. 2014;14(12):22313–41.CrossRefGoogle Scholar
  3. 3.
    Luppa PB, Bietenbeck A, Beaudoin C, Giannetti A. Clinically relevant analytical techniques, organizational concepts for application and future perspectives of point-of-care testing. Biotechnol Adv. 2016;34(3):139–60.CrossRefGoogle Scholar
  4. 4.
    Vashist SK, Schneider EM, Luong JHT. Commercial smartphone-based devices and smart applications for personalized healthcare monitoring and management. Diagnostics. 2014;4(3):104–28.CrossRefGoogle Scholar
  5. 5.
    Peacock PM, Zhang WJ, Trimpin S. Advances in ionization for mass spectrometry. Anal Chem. 2017;89(1):372–88.CrossRefGoogle Scholar
  6. 6.
    Vashist SK, Luppa PB, Yeo LY, Ozcan A, Luong JHT. Emerging technologies for next-generation point-of-care testing. Trends Biotechnol. 2015;33(11):692–705.CrossRefGoogle Scholar
  7. 7.
    Gauglitz G. Point-of-care platforms. Annu Rev Anal Chem. 2014;7:297–315.CrossRefGoogle Scholar
  8. 8.
    Vashist SK, Mudanyali O, Schneider EM, Zengerle R, Ozcan A. Cellphone-based devices for bioanalytical sciences. Anal Bioanal Chem. 2014;406(14):3263–77.CrossRefGoogle Scholar
  9. 9.
    Araz MK, Tentori AM, Herr AE. Microfluidic multiplexing in bioanalyses. J Lab Autom. 2013;18(5):350–66.CrossRefGoogle Scholar
  10. 10.
    Gordon J, Michel G. Discerning trends in multiplex immunoassay technology with potential for resource-limited settings. Clin Chem. 2012;58(4):690–8.CrossRefGoogle Scholar
  11. 11.
    Chin CD, Linder V, Sia SK. Commercialization of microfluidic point-of-care diagnostic devices. Lab Chip. 2012;12(12):2118–34.CrossRefGoogle Scholar
  12. 12.
    Rusling JF. Multiplexed electrochemical protein detection and translation to personalized cancer diagnostics. Anal Chem. 2013;85(11):5304–10.CrossRefGoogle Scholar
  13. 13.
    Dunbar SA. Applications of Luminex® xMAP™ technology for rapid, high-throughput multiplexed nucleic acid detection. Clin Chim Acta. 2006;363(1):71–82.CrossRefGoogle Scholar
  14. 14.
    Skogstrand K, Thorsen P, Norgaard-Pedersen B, Schendel DE, Sorensen LC, Hougaard DM. Simultaneous measurement of 25 inflammatory markers and neurotrophins in neonatal dried blood spots by immunoassay with xMAP technology. Clin Chem. 2005;51(10):1854–66.CrossRefGoogle Scholar
  15. 15.
    Kofoed K, Schneider UV, Scheel T, Andersen O, Eugen-Olsen J. Development and validation of a multiplex add-on assay for sepsis biomarkers using xMAP technology. Clin Chem. 2006;52(7):1284–93.CrossRefGoogle Scholar
  16. 16.
    Braeckmans K, De Smedt SC, Leblans M, Pauwels R, Demeester J. Encoding microcarriers: present and future technologies. Nat Rev Drug Discov. 2002;1(6):447–56.CrossRefGoogle Scholar
  17. 17.
    Ateya DA, Erickson JS, Howell PB Jr, Hilliard LR, Golden JP, Ligler FS. The good, the bad, and the tiny: a review of microflow cytometry. Anal Bioanal Chem. 2008;391(5):1485–98.CrossRefGoogle Scholar
  18. 18.
    Godin J, Chen CH, Cho SH, Qiao W, Tsai F, Lo YH. Microfluidics and photonics for bio-system-on-a-Chip: a review of advancements in technology towards a microfluidic flow cytometry chip. J Biophotonics. 2008;1(5):355–76.CrossRefGoogle Scholar
  19. 19.
  20. 20.
    Chowdhury F, Williams A, Johnson P. Validation and comparison of two multiplex technologies, Luminex® and Mesoscale discovery, for human cytokine profiling. J Immunol Methods. 2009;340(1):55–64.CrossRefGoogle Scholar
  21. 21.
    Fu Q, Zhu J, Van Eyk JE. Comparison of multiplex immunoassay platforms. Clin Chem. 2010;56(2):314–8.CrossRefGoogle Scholar
  22. 22.
    Breen EC, Reynolds SM, Cox C, Jacobson LP, Magpantay L, Mulder CB, et al. Multisite comparison of high-sensitivity multiplex cytokine assays. Clin Vaccine Immunol. 2011;18(8):1229–42.CrossRefGoogle Scholar
  23. 23.
    The EUROLINE: a new technique for extensive antibody profiles. 2017.
  24. 24.
  25. 25.
  26. 26.
    Clark TJ, McPherson PH, Buechler KF. The triage cardiac panel: cardiac markers for the triage system. Point of Care. 2002;1(1):42–6.Google Scholar
  27. 27.
  28. 28.
  29. 29.
  30. 30.
  31. 31.
    Biochip immunoassays. 2018.
  32. 32.
    Multiplex testing. 2018.
  33. 33.
  34. 34.
  35. 35.
  36. 36.
  37. 37.
  38. 38.
  39. 39.
    Gorkin R, Park J, Siegrist J, Amasia M, Lee BS, Park JM, et al. Centrifugal microfluidics for biomedical applications. Lab Chip. 2010;10(14):1758–73.CrossRefGoogle Scholar
  40. 40.
  41. 41.
    Sackmann EK, Fulton AL, Beebe DJ. The present and future role of microfluidics in biomedical research. Nature. 2014;507(7491):181–9.CrossRefGoogle Scholar
  42. 42.
    Robinson T, Dittrich PS. Microfluidic technology for molecular diagnostics. Adv Biochem Eng Biotechnol. 2013;133:89–114.Google Scholar
  43. 43.
    Feng LN, Bian ZP, Peng J, Jiang F, Yang GH, Zhu YD, et al. Ultrasensitive multianalyte electrochemical immunoassay based on metal ion functionalized titanium phosphate nanospheres. Anal Chem. 2012;84(18):7810–5.CrossRefGoogle Scholar
  44. 44.
    Kong F-Y, Xu B-Y, Xu J-J, Chen HY. Simultaneous electrochemical immunoassay using CdS/DNA and PbS/DNA nanochains as labels. Biosens Bioelectron. 2012;39(1):177–82.CrossRefGoogle Scholar
  45. 45.
    Wang J, Liu G, Merkoci A. Electrochemical coding technology for simultaneous detection of multiple DNA targets. J Am Chem Soc. 2003;125(11):3214–5.CrossRefGoogle Scholar
  46. 46.
    Tang D, Hou L, Niessner R, Xu M, Gao Z, Knopp DJB, et al. Multiplexed electrochemical immunoassay of biomarkers using metal sulfide quantum dot nanolabels and trifunctionalized magnetic beads. Biosens Bioelectron. 2013;46:37–43.CrossRefGoogle Scholar
  47. 47.
    Tang J, Tang D, Niessner R, Chen G, Knopp D. Magneto-controlled graphene immunosensing platform for simultaneous multiplexed electrochemical immunoassay using distinguishable signal tags. Anal Chem. 2011;83(13):5407–14.CrossRefGoogle Scholar
  48. 48.
    Sato K, Yamanaka M, Takahashi H, Tokeshi M, Kimura H, Kitamori T. Microchip-based immunoassay system with branching multichannels for simultaneous determination of interferon-gamma. Electrophoresis. 2002;23(5):734–9.CrossRefGoogle Scholar
  49. 49.
    Ko YJ, Maeng JH, Ahn Y, Hwang SY, Cho NG, Lee SH. Microchip-based multiplex electro-immunosensing system for the detection of cancer biomarkers. Electrophoresis. 2008;29(16):3466–76.CrossRefGoogle Scholar
  50. 50.
    Shriver-Lake LC, Golden J, Bracaglia L, Ligler FS. Simultaneous assay for ten bacteria and toxins in spiked clinical samples using a microflow cytometer. Anal Bioanal Chem. 2013;405(16):5611–4.CrossRefGoogle Scholar
  51. 51.
    Hashemi N, Erickson JS, Golden JP, Ligler FS. Optofluidic characterization of marine algae using a microflow cytometer. Biomicrofluidics. 2011;5(3):032009.CrossRefGoogle Scholar
  52. 52.
    Vashist SK, Luong JHT. Handbook of immunoassay technologies: approaches, performances, and applications. London: Academic Press; 2018.Google Scholar
  53. 53.
    Li J, Macdonald J. Multiplexed lateral flow biosensors: technological advances for radically improving point-of-care diagnoses. Biosens Bioelectron. 2016;83:177–92.CrossRefGoogle Scholar
  54. 54.
    Li J, Macdonald J. Multiplex lateral flow detection and binary encoding enables a molecular colorimetric 7-segment display. Lab Chip. 2016;16(2):242–5.CrossRefGoogle Scholar
  55. 55.
    Song S, Liu N, Zhao Z, Njumbe Ediage E, Wu S, Sun C, et al. Multiplex lateral flow immunoassay for mycotoxin determination. Anal Chem. 2014;86(10):4995–5001.CrossRefGoogle Scholar
  56. 56.
    Taranova N, Berlina A, Zherdev A, Dzantiev BJB. ‘Traffic light’immunochromatographic test based on multicolor quantum dots for the simultaneous detection of several antibiotics in milk. Biosens Bioelectron. 2015;63:255–61.CrossRefGoogle Scholar
  57. 57.
    Lafleur LK, Bishop JD, Heiniger EK, Gallagher RP, Wheeler MD, Kauffman P, et al. A rapid, instrument-free, sample-to-result nucleic acid amplification test. Lab Chip. 2016;16(19):3777–87.CrossRefGoogle Scholar
  58. 58.
    Mao X, Baloda M, Gurung AS, Lin Y, Liu G. Multiplex electrochemical immunoassay using gold nanoparticle probes and immunochromatographic strips. Electrochem Commun. 2008;10(10):1636–40.CrossRefGoogle Scholar
  59. 59.
    Mao X, Wang W, Du T-E. Rapid quantitative immunochromatographic strip for multiple proteins test. Sens Actuators B: Chemical. 2013;186:315–20.CrossRefGoogle Scholar
  60. 60.
  61. 61.
    Ahmed S, Bui MP, Abbas A. Paper-based chemical and biological sensors: engineering aspects. Biosens Bioelectron. 2016;77:249–63.CrossRefGoogle Scholar
  62. 62.
    Rolland JP, Mourey DA. Paper as a novel material platform for devices. MRS Bull. 2013;38(4):299–305.CrossRefGoogle Scholar
  63. 63.
    Yang Y, Noviana E, Nguyen MP, Geiss BJ, Dandy DS, Henry CS. Paper-based microfluidic devices: emerging themes and applications. Anal Chem. 2017;89(1):71–91.CrossRefGoogle Scholar
  64. 64.
    Vella SJ, Beattie P, Cademartiri R, Laromaine A, Martinez AW, Phillips ST, et al. Measuring markers of liver function using a micropatterned paper device designed for blood from a fingerstick. Anal Chem. 2012;84(6):2883–91.CrossRefGoogle Scholar
  65. 65.
    Pollock NR, Rolland JP, Kumar S, Beattie PD, Jain S, Noubary F, et al. A paper-based multiplexed transaminase test for low-cost, point-of-care liver function testing. Sci Transl Med. 2012;4(152):152ra29.CrossRefGoogle Scholar
  66. 66.
    Dungchai W, Chailapakul O, Henry CS. Electrochemical detection for paper-based microfluidics. Anal Chem. 2009;81(14):5821–6.CrossRefGoogle Scholar
  67. 67.
    Ge L, Yan J, Song X, Yan M, Ge S, Yu J. Three-dimensional paper-based electrochemiluminescence immunodevice for multiplexed measurement of biomarkers and point-of-care testing. Biomaterials. 2012;33(4):1024–31.CrossRefGoogle Scholar
  68. 68.
    Li X, Liu X. A microfluidic paper-based origami nanobiosensor for label-free, ultrasensitive immunoassays. Adv Healthc Mater. 2016;5(11):1326–35.CrossRefGoogle Scholar
  69. 69.
    Li W, Li L, Ge S, Song X, Ge L, Yan M, et al. Multiplex electrochemical origami immunodevice based on cuboid silver-paper electrode and metal ions tagged nanoporous silver–chitosan. Biosens Bioelectron. 2014;56:167–73.CrossRefGoogle Scholar
  70. 70.
    Wu Y, Xue P, Hui KM, Kang Y. A paper-based microfluidic electrochemical immunodevice integrated with amplification-by-polymerization for the ultrasensitive multiplexed detection of cancer biomarkers. Biosens Bioelectron. 2014;52:180–7.CrossRefGoogle Scholar
  71. 71.
    Wu Y, Xue P, Kang Y, Hui KM. Paper-based microfluidic electrochemical immunodevice integrated with nanobioprobes onto graphene film for ultrasensitive multiplexed detection of cancer biomarkers. Anal Chem. 2013;85(18):8661–8.CrossRefGoogle Scholar
  72. 72.
    Zang D, Ge L, Yan M, Song X, Yu J. Electrochemical immunoassay on a 3D microfluidic paper-based device. Chem Commun. 2012;48(39):4683–5.CrossRefGoogle Scholar
  73. 73.
    Vashist SK, Lam E, Hrapovic S, Male KB, Luong JHT. Immobilization of antibodies and enzymes on 3-aminopropyltriethoxysilane-functionalized bioanalytical platforms for biosensors and diagnostics. Chem Rev. 2014;114(21):11083–130.CrossRefGoogle Scholar
  74. 74.
    Ling MM, Ricks C, Lea P. Multiplexing molecular diagnostics and immunoassays using emerging microarray technologies. Expert Rev Mol Diagn. 2007;7(1):87–98.CrossRefGoogle Scholar
  75. 75.
    Chandra PE, Sokolove J, Hipp BG, Lindstrom TM, Elder JT, Reveille JD, et al. Novel multiplex technology for diagnostic characterization of rheumatoid arthritis. Arthritis Res Ther. 2011;13(3):R102.CrossRefGoogle Scholar
  76. 76.
    Kadimisetty K, Malla S, Sardesai NP, Joshi AA, Faria RC, Lee NH, et al. Automated multiplexed ECL Immunoarrays for cancer biomarker proteins. Anal Chem. 2015;87(8):4472–8.CrossRefGoogle Scholar
  77. 77.
    Chen P, Chung MT, McHugh W, Nidetz R, Li Y, Fu J, et al. Multiplex serum cytokine immunoassay using nanoplasmonic biosensor microarrays. ACS Nano. 2015;9(4):4173–81.CrossRefGoogle Scholar
  78. 78.
    Masson JF. Surface plasmon resonance clinical biosensors for medical diagnostics. ACS Sens. 2017;2(1):16–30.CrossRefGoogle Scholar
  79. 79.
    Acimovic SS, Ortega MA, Sanz V, Berthelot J, Garcia-Cordero JL, Renger J, et al. LSPR chip for parallel, rapid, and sensitive detection of cancer markers in serum. Nano Lett. 2014;14(5):2636–41.CrossRefGoogle Scholar
  80. 80.
    Schumacher S, Nestler J, Otto T, Wegener M, Ehrentreich-Forster E, Michel D, et al. Highly-integrated lab-on-chip system for point-of-care multiparameter analysis. Lab Chip. 2012;12(3):464–73.CrossRefGoogle Scholar
  81. 81.
    Otieno BA, Krause CE, Jones AL, Kremer RB, Rusling JF. Cancer diagnostics via ultrasensitive multiplexed detection of parathyroid hormone-related peptides with a microfluidic immunoarray. Anal Chem. 2016;88(18):9269–75.CrossRefGoogle Scholar
  82. 82.
    Wilson MS, Nie W. Multiplex measurement of seven tumor markers using an electrochemical protein chip. Anal Chem. 2006;78(18):6476–83.CrossRefGoogle Scholar
  83. 83.
    Wan Y, Su Y, Zhu X, Liu G, Fan C. Development of electrochemical immunosensors towards point of care diagnostics. Biosens Bioelectron. 2013;47:1–11.CrossRefGoogle Scholar
  84. 84.
    Díaz-González M, Muñoz-Berbel X, Jiménez-Jorquera C, Baldi A, Fernández-Sánchez C. Diagnostics using multiplexed electrochemical readout devices. Electroanalysis. 2014;26(6):1154–70.CrossRefGoogle Scholar
  85. 85.
    Ghindilis AL, Smith MW, Schwarzkopf KR, Roth KM, Peyvan K, Munro SB, et al. CombiMatrix oligonucleotide arrays: genotyping and gene expression assays employing electrochemical detection. Biosens Bioelectron. 2007;22(9–10):1853–60.CrossRefGoogle Scholar
  86. 86.
    Roth KM, Peyvan K, Schwarzkopf KR, Ghindilis A. Electrochemical detection of short DNA oligomer hybridization using the CombiMatrix ElectraSense microarray reader. Electroanalysis. 2006;18(19–20):1982–8.CrossRefGoogle Scholar
  87. 87.
    Karle M, Vashist SK, Zengerle R, von Stetten F. Microfluidic solutions enabling continuous processing and monitoring of biological samples: a review. Anal Chim Acta. 2016;929:1–22.CrossRefGoogle Scholar
  88. 88.
    Duncan PN, Ahrar S, Hui EE. Scaling of pneumatic digital logic circuits. Lab Chip. 2015;15(5):1360–5.CrossRefGoogle Scholar
  89. 89.
    Araci IE, Brisk P. Recent developments in microfluidic large scale integration. Curr Opin Biotechnol. 2014;25:60–8.CrossRefGoogle Scholar
  90. 90.
    Shao H, Chung J, Lee K, Balaj L, Min C, Carter BS, et al. Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma. Nat Commun. 2015;6:6999.CrossRefGoogle Scholar
  91. 91.
    Chin CD, Laksanasopin T, Cheung YK, Steinmiller D, Linder V, Parsa H, et al. Microfluidics-based diagnostics of infectious diseases in the developing world. Nat Med. 2011;17(8):1015–9.CrossRefGoogle Scholar
  92. 92.
    Lafleur L, Stevens D, McKenzie K, Ramachandran S, Spicar-Mihalic P, Singhal M, et al. Progress toward multiplexed sample-to-result detection in low resource settings using microfluidic immunoassay cards. Lab Chip. 2012;12(6):1119–27.CrossRefGoogle Scholar
  93. 93.
    Kling A, Chatelle C, Armbrecht L, Qelibari E, Kieninger J, Dincer C, et al. Multianalyte antibiotic detection on an electrochemical microfluidic platform. Anal Chem. 2016;88(20):10036–43.CrossRefGoogle Scholar
  94. 94.
    Vashist SK, Luong JHT. Bioanalytical requirements and regulatory guidelines for immunoassays. In: Handbook of immunoassay technologies. London: Elsevier; 2018. p. 81–95.CrossRefGoogle Scholar
  95. 95.
    Vashist SK, Luong JHT. Trends in in vitro diagnostics and mobile healthcare. Biotechnol Adv. 2016;34(3):137–8.CrossRefGoogle Scholar
  96. 96.
    Contreras-Naranjo JC, Wei Q, Ozcan A. Mobile phone-based microscopy, sensing, and diagnostics. IEEE J Sel Top Quantum Electron. 2016;22(3):1–14.CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Sandeep Kumar Vashist
    • 1
  1. 1.Labsystems Diagnostics OyVantaaFinland

Personalised recommendations