Colorimetric Sandwich Assays for Protein Detection

  • Xiaoqing Yi
  • Rui Liu
  • Xiaoding LouEmail author
  • Fan Xia


For decades, the sandwich assays have been widely used in quality control, clinical diagnostics, biological detection, and environmental monitoring field. Apart from the requirement of labeling the molecular target, the sandwich assay generally requires the recognition and signaling probe to be combined, which gives them accurate specific. Also, the optical sensing method is of great interest due to the intrinsic high sensitivity and simplicity. The colorimetric assays are much simpler for the detection of analytes, and their response can be directly detected with bare eyes or by photometry compared with other analytical techniques. Colorimetric sandwich assays are usually based on the observable color variation in the presence of enzyme-labeled antibody. In this chapter, we focus on the detection of protein by colorimetric sandwich assays based on traditional enzymes and biomimetic nanomaterials. The detection technologies employed in colorimetric sandwich assays based on traditional enzymes were alkaline phosphatase (ALP), horseradish peroxidase (HRP), open sandwich immunoassay (OS-IA), and gold–multienzyme–nanocarrier. For the biomimetic nanomaterials, highlighted examples were focused on Fe3O4 magnetic nanoparticles (MNPs) biomimetic enzymes, and Au@Pt nanostructure biomimetic enzymes.


Colorimetric sandwich assays Protein detection Biomimetic enzymes Traditional enzymes Nanoparticles 


  1. 1.
    Liu YX, Huang XG, Ren JC (2016) Recent advances in chemiluminescence detection coupled with capillary electrophoresis and microchip capillary electrophoresis. Electrophoresis 37:2–18CrossRefGoogle Scholar
  2. 2.
    Klapkova E, Fortova M, Prusa R, Moravcova L, Kotaska K (2016) Determination of urine albumin by new simple high-performanceliquid chromatography method. J Clin Lab Anal 30:1226–1231CrossRefGoogle Scholar
  3. 3.
    Doussineau T, Mathevon C, Altamura L, Vendrely C, Dugourd P, Forge V, Antoine R (2016) Mass determination of entire amyloid fibrils by using mass spectrometry. Angew Chem Int Ed 55:2340–2344CrossRefGoogle Scholar
  4. 4.
    Engvall E, Perlmann P (1971) Enzyme-linked immunosorbent assay (ELISA). Quant Assay Immunoglobulin G. Immunochem 8:871–874Google Scholar
  5. 5.
    Tomita H, Ogawa M, Kamijo T, Mori O, Ishikawa E, Mohri Z, Murakami Y (1989) A highly sensitive sandwich enzyme immunoassay of urinary growth hormone in children with short stature, Turner’s syndrome, and simple obesity. Acta Endocrinol 121:513–519CrossRefGoogle Scholar
  6. 6.
    Li W, Qiang WB, Li J, Li H, Dong YF, Zhao YJ, Xu DK (2014) Nanoparticle-catalyzed reductive bleaching for fabricating turn-off and enzyme-free amplified colorimetric bioassays. Biosens Bioelectron 51:219–224CrossRefGoogle Scholar
  7. 7.
    Arya SK, Bhansali S (2011) Lung cancer and its early detection using biomarker-based biosensors. Chem Rev 111:6783–6809CrossRefGoogle Scholar
  8. 8.
    Lei JQ, Jing T, Zhou TT, Zhou YS, Wu W, Mei SR, Zhou YK (2014) A simple and sensitive immunoassay for the determination of human chorionic gonadotropin by graphene-based chemiluminescence resonance energy transfer. Biosens Bioelectron 54:72–77CrossRefGoogle Scholar
  9. 9.
    Ke RQ, Yang W, Xia XH, Xu Y, Li QG (2010) Tandem conjugation of enzyme and antibody on silica nanoparticle for enzyme immunoassay. Anal Biochem 406:8–13CrossRefGoogle Scholar
  10. 10.
    Ambrosi A, Castaneda MT, Killard AJ, Smyth MR, Alegret S, Merkoci A (2007) Double-codified gold nanolabels for enhanced immunoanalysis. Anal Chem 79:5232–5240CrossRefGoogle Scholar
  11. 11.
    Liu MY, Jia CP, Huang YY, Lou XH, Yao SH, Jin QH, Zhao JL, Xiang JQ (2010) Highly sensitive protein detection using enzyme-labeled gold nanoparticle probes. Analyst 135:327–331CrossRefGoogle Scholar
  12. 12.
    Shen JW, Li YB, Gu HS, Xia F, Zuo XL (2014) Recent development of sandwich assay based on the nanobiotechnologies for proteins, nucleic acids, small molecules, and Ions. Chem Rev 114:7631–7677CrossRefGoogle Scholar
  13. 13.
    Gao LZ, Zhuang J, Nie L, Zhang JB, Zhang Y, Gu N, Wang TH, Feng J, Yang DL, Perrett S, Yan XY (2007) Intrinsic peroxidase-like activity of ferromagnetic nanoparticles. Nat Nanotechnol 2:577–583CrossRefGoogle Scholar
  14. 14.
    Wei H, Wang E (2013) Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes. Chem Soc Rev 42:6060–6093CrossRefGoogle Scholar
  15. 15.
    Song YJ, Wang XH, Zhao C, Qu KG, Ren JS, Qu XG (2010) Label-free colorimetric detection of single nucleotide polymorphism by using single-walled carbon nanotube intrinsic peroxidase-like activity. Chem—Eur J 16:3617–3621Google Scholar
  16. 16.
    Dai ZH, Liu SH, Bao JC, Jui HC (2009) Nanostructured FeS as a mimic peroxidase for biocatalysis and biosensing. Chem—Eur J 15:4321–4326Google Scholar
  17. 17.
    Chen W, Chen J, Liu AL, Wang LM, Li GW, Lin XH (2011) Peroxidase-like activity of cupric oxide nanoparticle. ChemCatChem 3:1151–1154CrossRefGoogle Scholar
  18. 18.
    Cleland WW, Hengge AC (2006) Enzymatic mechanisms of phosphate and sulfate transfer. Chem Rev 106:3252–3278CrossRefGoogle Scholar
  19. 19.
    Ko YC, Mukaida N, Panyutich A, Voitenok NN, Matsushima K, Kawai T, Kasahara T (1992) A sensitive enzyme-linked-immunosorbent-assay for human interleukin-8. J Immunol Methods 149:227–235CrossRefGoogle Scholar
  20. 20.
    Daniilidou M, Tsolaki M, Giannakouros T, Nikolakaki E (2011) Detection of elevated antibodies against SR protein kinase 1 in the serum of Alzheimer’s disease patients. J Neuroimmunol 238:67–72CrossRefGoogle Scholar
  21. 21.
    Vandermeeren M, Mercken M, Vanmechelen E, Six J, Vandevoorde A, Martin JJ, Cras P (1993) Detection of tau proteins in normal and alzheimers-disease cerebrospinal-fluid with a sensitive sandwich enzyme-linked-immunosorbent-assay. J Neurochem 61:1828–1834CrossRefGoogle Scholar
  22. 22.
    Surojanametakul V, Doi H, Shibata H, Mizumura T, Takahashi T, Varanyanond W, Wannapinpong S, Shoji M, Ito T, Tamura H (2011) Reliable enzyme-linked immunosorbent assay for the determination of coconut milk proteins in processed foods. J Agric Food Chem 59:2131–2136Google Scholar
  23. 23.
    Mercken M, Vandermeeren M, Lubke U, Six J, Boons J, Vanmechelen E, Vandevoorde A, Gheuens J (1992) Affinity purification of human tau-proteins and the construction of a sensitive enzyme-linked-immunosorbent-assay for tau-detection. J Neurochem 58:548–553CrossRefGoogle Scholar
  24. 24.
    Frey A, Meckelein B, Externest D, Schmidt MA (2000) A stable and highly sensitive 3, 3’, 5, 5’-tetramethylbenzidine-based substrate reagent for enzyme-linked immunosorbent assays. J Immunol Methods 233:47–56CrossRefGoogle Scholar
  25. 25.
    El Mouedden M, Vandermeeren M, Meert T, Mercken M (2005) Development of a specific ELISA for the quantitative study of amino-terminally truncated beta-amyloid peptides. J Neurosci Methods 145:97–105CrossRefGoogle Scholar
  26. 26.
    Castigliego L, Li XN, Armani A, Mazzi M, Guidi A (2011) An immunoenzymatic assay to measure insulin-like growth factor 1 (IGF-1) in buffalo milk with an IGF binding protein blocking pre-treatment of the sample. Int Dairy J 21:421–426CrossRefGoogle Scholar
  27. 27.
    Wang J, Cao Y, Xu YY, Li GX (2009) Colorimetric multiplexed immunoassay for sequential detection of tumor markers. Biosens Bioelectron 25:532–536CrossRefGoogle Scholar
  28. 28.
    Park JH, Cho YS, Kang S, Lee EJ, Lee GH, Hah SS (2014) A colorimetric sandwich-type assay for sensitive thrombin detection based on enzyme-linked aptamer assay. Anal Biochem 462:10–12CrossRefGoogle Scholar
  29. 29.
    Abuknesha RA, Jeganathan F, DeGroot R, Wildeboer D, Price RG (2010) Detection of proteases using an immunochemical method with haptenylated-gelatin as a solid-phase substrate. Anal Bioanal Chem 396:2547–2558CrossRefGoogle Scholar
  30. 30.
    Gehring A, He XH, Fratamico P, Lee J, Bagi L, Brewster J, Paoli G, He YP, Xie YP, Skinner C, Barnett C, Harris D (2014) A high-throughput, precipitating colorimetric sandwich ELISA microarray for shiga toxins. Toxins 6:1855–1872CrossRefGoogle Scholar
  31. 31.
    Chen CH, Liu YF, Zheng ZH, Zhou GH, Ji XH, Wang HZ, He ZK (2015) A new colorimetric platform for ultrasensitive detection of protein and cancer cells based on the assembly of nucleic acids and proteins. Anal Chim Acta 880:1–7CrossRefGoogle Scholar
  32. 32.
    Islam KN, Ihara M, Dong JH, Kasagi N, Mori T, Ueda H (2011) Direct construction of an open-sandwich enzyme immunoassay for one-step noncompetitive detection of thyroid hormone T4. Anal Chem 83:1008–1014CrossRefGoogle Scholar
  33. 33.
    Suzuki C, Ueda H, Mahoney W, Nagamune T (2000) Open sandwich enzyme-linked immunosorbent assay for the quantitation of small haptens. Anal Biochem 286:238–246CrossRefGoogle Scholar
  34. 34.
    Aburatani T, Sakamoto K, Masuda K, Nishi K, Ohkawa H, Nagamune T, Ueda H (2003) A general method to select antibody fragments suitable for noncompetitive detection of monovalent antigens. Anal Chem 75:4057–4064CrossRefGoogle Scholar
  35. 35.
    Liu XB, Eichenberger M, Fujioka Y, Dong JH, Ueda H (2012) Improved detection sensitivity and selectivity attained by open-sandwich selection of an anti-estradiol antibody. Anal Sci 28:861–867CrossRefGoogle Scholar
  36. 36.
    Wang SS, Chen ZP, Choo J, Chen LX (2016) Naked-eye sensitive ELISA-like assay based on gold-enhanced peroxidase-like immunogold activity. Anal Bioanal Chem 408:1015–1022CrossRefGoogle Scholar
  37. 37.
    Zor E, Bekar N (2017) Lab-in-a-syringe using gold nanoparticles for rapid colorimetric chiral discrimination of enantiomers. Biosens Bioelectron 91:211–216CrossRefGoogle Scholar
  38. 38.
    Ambrosi A, Airo F, Merkoci A (2010) Enhanced gold nanoparticle based ELISA for a breast cancer biomarker. Anal Chem 82:1151–1156CrossRefGoogle Scholar
  39. 39.
    Tang SX, Hewlett I (2010) Nanoparticle-based immunoassays for sensitive and early detection of HIV-1 capsid (p24) antigen. J Infect Dis 201:S59–S64CrossRefGoogle Scholar
  40. 40.
    Zhang ZY, Chen ZP, Wang SS, Cheng FB, Chen LX (2015) Iodine-mediated etching of gold nanorods for plasmonic ELISA based on colorimetric detection of alkaline phosphatase. ACS Appl Mater Interfaces 7:27639–27645CrossRefGoogle Scholar
  41. 41.
    Dixit CK, Vashist SK, O’Neill FT, O’Reilly B, MacCraith BD, O’Kennedy R (2010) Development of a high sensitivity rapid sandwich ELISA procedure and its comparison with the conventional approach. Anal Chem 82:7049–7052CrossRefGoogle Scholar
  42. 42.
    Song YJ, Qu KG, Zhao C, Ren JS, Qu XG (2010) Graphene oxide: intrinsic peroxidase catalytic activity and its application to glucose detection. Adv Mater 22:2206–2210CrossRefGoogle Scholar
  43. 43.
    He WW, Liu Y, Yuan JS, Yin JL, Wu XC, Hu XN, Zhang K, Liu JB, Chen CY, Ji YL, Guo YT (2011) Au@Pt nanostructures as oxidase and peroxidase mimetics for use in immunoassays. Biomaterials 32:1139–1147CrossRefGoogle Scholar
  44. 44.
    Ma M, Zhang Y, Gu N (2011) Peroxidase-like catalytic activity of cubic Pt nanocrystals. Colloids Surf A Physicochem Eng Asp 373:6–10CrossRefGoogle Scholar
  45. 45.
    Song P, Wang Q, Zhang Z, Yang ZX (2010) Synthesis and gas sensing properties of biomorphic LaFeO3 hollow fibers templated from cotton. Sens Actuators, B 147:248–254CrossRefGoogle Scholar
  46. 46.
    Gao ZQ, Xu MD, Lu MH, Chen GN, Tang DP (2015) Urchin-like (gold core)@(platinum shell) nanohybrids: a highly efficient peroxidase-mimetic system for in situ amplified colorimetric immunoassay. Biosens Bioelectron 70:194–201CrossRefGoogle Scholar
  47. 47.
    Zheng C, Zheng AX, Liu B, Zhang XL, He Y, Li J, Yang HH, Chen GN (2014) One-pot synthesized DNA-templated Ag/Pt bimetallic nanoclusters as peroxidase mimics for colorimetric detection of thrombin. Chem Commun 50:13103–13106CrossRefGoogle Scholar

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© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  1. 1.Engineering Research Center of Nano-Geomaterials of Ministry of Education, Faculty of Materials Science and ChemistryChina University of GeosciencesWuhanPeople’s Republic of China
  2. 2.Hubei Key Laboratory of Bioinorganic Chemistry & Materia Medica, School of Chemistry and Chemical EngineeringHuazhong University of Science and TechnologyWuhanPeople’s Republic of China

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