Fluorescence Sandwich Assays for Protein Detection

  • Fujian HuangEmail author
  • Fan Xia


The detection of protein with high sensitivity and selectivity is of great important for protein fundamental functions study and clinic diagnostics. Sandwich assays have been developed for multivalent proteins detection and have been prevailing for decades in the field of clinical diagnostics and bio-detection. The sandwich assays usually give a high sensitivity and selectivity because of the usage of a couple of match recognition probe and signal probe. This chapter summarizes recent advances in the sandwich assays for protein detections with fluorescence as signal outputs. Different recognition or signal elements such as antibodies and aptamers and fluorescence signal reporters (organic dyes, nanomaterials, and conjugated polymers) are discussed in details in this chapter.


Protein detection Fluorescence signal reporter Sandwich assay Aptamer Antibody 


  1. 1.
    Vasan RS (2006) Biomarkers of cardiovascular disease—molecular basis and practical considerations. Circulation 113:2335–2362CrossRefGoogle Scholar
  2. 2.
    Molitoris BA, Melnikov VY, Okusa MD, Hirnmelfarb J (2008) Technology insight: biomarker development in acute kidney injury—what can we anticipate? Nat Clin Pract Nephr 4:154–165CrossRefGoogle Scholar
  3. 3.
    Bilitewski U (2006) Protein-sensing assay formats and devices. Anal Chim Acta 568:232–247CrossRefGoogle Scholar
  4. 4.
    Chen AL, Yan MM, Yang SM (2016) Split aptamers and their applications in sandwich aptasensors. TrAC Trends Anal Chem 80:581–593CrossRefGoogle Scholar
  5. 5.
    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
  6. 6.
    Shankar G, Devanarayan V, Amaravadi L, Barrett YC, Bowsher R, Finco-Kent D, Fiscella M, Gorovits B, Kirschner S, Moxness M, Parish T, Quarmby V, Smith H, Smith W, Zuckerman LA, Koren E (2008) Recommendations for the validation of immunoassays used for detection of host antibodies against biotechnology products. J Pharm Biomed Anal 48:1267–1281CrossRefGoogle Scholar
  7. 7.
    Morgan CL, Newman DJ, Price CP (1996) Immunosensors: technology and opportunities in laboratory medicine. Clin Chem 42:193–209Google Scholar
  8. 8.
    Hock B (1997) Antibodies for immunosensors—a review. Anal Chim Acta 347:177–186CrossRefGoogle Scholar
  9. 9.
    Vanderlugt CL, Miller SD (2002) Epitope spreading in immunemediated diseases: implications for immunotherapy. Nat Rev Immunol 2:85–95CrossRefGoogle Scholar
  10. 10.
    Kiening M, Niessner R, Weller MG (2005) Microplate-based screening methods for the efficient development of sandwich immunoassays. Analyst 130:1580–1588CrossRefGoogle Scholar
  11. 11.
    Stoevesandt O, Taussig MJ (2007) Affinity reagent resources for human proteome detection: Initiatives and perspectives. Proteomics 7:2738–2750CrossRefGoogle Scholar
  12. 12.
    Anderson NL, Anderson NG, Pearson TW, Borchers CH, Paulovich AG, Patterson SD, Gillette M, Aebersold R, Carr SA (2009) A human proteome detection and quantitation project. Mol Cell Proteomics 8:883–886CrossRefGoogle Scholar
  13. 13.
    Toh SY, Citartan M, Gopinath SCB, Tang T-H (2015) Aptamers as a replacement for antibodies in enzyme-linked immunosorbent assay. Biosens Bioelectron 64:392–403CrossRefGoogle Scholar
  14. 14.
    Jayasena SD (1999) Aptamers: an emerging class of molecules that rival antibodies in diagnostics. Clin Chem 45:1628–1650Google Scholar
  15. 15.
    Lofblom J, Feldwisch J, Tolmachev V, Carlsson J, Stahl S, Frejd FY (2010) Affibody molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications. FEBS Lett 584:2670–2680CrossRefGoogle Scholar
  16. 16.
    Ellington AD, Szostak JW (1990) Invitro selection of RNA molecules that bind specific ligands. Nature 346:818–822CrossRefGoogle Scholar
  17. 17.
    Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505–510CrossRefGoogle Scholar
  18. 18.
    Kawano R, Osaki T, Sasaki H, Takinoue M, Yoshizawa S, Takeuchi S (2011) Rapid detection of a cocaine-binding aptamer using biological nanopores on a chip. J Am Chem Soc 133:8474–8477CrossRefGoogle Scholar
  19. 19.
    Zhang HQ, Wang ZW, Li XF, Le XC (2006) Ultrasensitive detection of proteins by amplification of affinity aptamers. Angew Chem Int Ed 45:1576–1580CrossRefGoogle Scholar
  20. 20.
    Shangguan DH, Li Y, Tang ZW, Cao ZC, Chen HW, Mallikaratchy P, Sefah K, Yang CJ, Tan WH (2006) Aptamers evolved from live cells as effective molecular probes for cancer study. Proc Natl Acad Sci U S A 103:11838–11843CrossRefGoogle Scholar
  21. 21.
    Zhang LQ, Wan S, Jiang Y, Wang YY, Fu T, Liu QL, Cao ZJ, Qiu LP, Tan WH (2017) Molecular elucidation of disease biomarkers at the interface of chemistry and biology. J Am Chem Soc 139:2532–2540CrossRefGoogle Scholar
  22. 22.
    Justino CIL, Freitas AC, Pereira R, Duarte AC, Santos TAPR (2015) Recent developments in recognition elements for chemical sensors and biosensors. TrAC Trends Anal Chem 68:2–17CrossRefGoogle Scholar
  23. 23.
    Bunka DHJ, Stockley PG (2006) Aptamers come of age—at last. Nat Rev Microbiol 4:588–596CrossRefGoogle Scholar
  24. 24.
    Tombelli S, Minunni M, Mascini A (2005) Analytical applications of aptamers. Biosens Bioelectron 20:2424–2434CrossRefGoogle Scholar
  25. 25.
    Li JJ, Zhong XQ, Zhang HQ, Le XC, Zhu JJ (2012) Binding-induced fluorescence turn-on assay using aptamer-functionalized silver nanocluster DNA probes. Anal Chem 84:5170–5174CrossRefGoogle Scholar
  26. 26.
    Tokunaga T, Namiki S, Yamada K, Imaishi T, Nonaka H, Hirose K, Sando S (2012) Cell surface-anchored fuorescent aptamer sensor enables imaging of chemical transmitter dynamics. J Am Chem Soc 134:9561–9564CrossRefGoogle Scholar
  27. 27.
    Gopinath SCB, Lakshmipriya T, Awazu K (2014) Colorimetric detection of controlled assembly and disassembly of aptamers on unmodified gold nanoparticles. Biosens Bioelectron 51:115–123CrossRefGoogle Scholar
  28. 28.
    Kim YS, Kim JH, Kim IA, Lee SJ, Gu MB (2011) The affinity ratio-Its pivotal role in gold nanoparticle-based competitive colorimetric aptasensor. Biosens Bioelectron 26:4058–4063CrossRefGoogle Scholar
  29. 29.
    Li SY, Chen DY, Zhou QT, Wang W, Gao LF, Jiang J, Liang HJ, Liu YZ, Liang GL, Cui H (2014) A general chemiluminescence strategy for measuring aptamer-target binding and target concentration. Anal Chem 86:5559–5566CrossRefGoogle Scholar
  30. 30.
    Xie LP, Yan XJ, Du YN (2014) An aptamer based wall-less LSPR array chip for label-free and high throughput detection of biomolecules. Biosens Bioelectron 53:58–64CrossRefGoogle Scholar
  31. 31.
    Ashley J, Li SFY (2013) An aptamer based surface plasmon resonance biosensor for the detection of bovine catalase in milk. Biosens Bioelectron 48:126–131CrossRefGoogle Scholar
  32. 32.
    Labib M, Zamay AS, Kolovskaya OS, Reshetneva IT, Zamay GS, Kibbee RJ, Sattar SA, Zamay TN, Berezovski MV (2012) Aptamer-based impedimetric sensor for bacterial typing. Anal Chem 84:8114–8117CrossRefGoogle Scholar
  33. 33.
    Yang ZG, Kasprzyk-Hordern B, Goggins S, Frost CG, Estrela P (2015) A novel immobilization strategy for electrochemical detection of cancer biomarkers: DNA-directed immobilization of aptamer sensors for sensitive detection of prostate specific antigens. Analyst 140:2628–2633CrossRefGoogle Scholar
  34. 34.
    Pan L, Huang Y, Wen CC, Zhao SL (2013) Label-free fluorescence probe based on structure-switching aptamer for the detection of interferon gamma. Analyst 138:6811–6816CrossRefGoogle Scholar
  35. 35.
    Stojanovic MN, de Prada P, Landry DW (2000) Fluorescent sensors based on aptamer self-assembly. J Am Chem Soc 122:11547–11548CrossRefGoogle Scholar
  36. 36.
    Yue QL, Shen T, Wang L, Xu SL, Li HB, Xue QW, Zhang YF, Gu XH, Zhang SQ, Liu JF (2014) A convenient sandwich assay of thrombin in biological media using nanoparticle-enhanced fluorescence polarization. Biosens Bioelectron 56:231–236CrossRefGoogle Scholar
  37. 37.
    Tennico YH, Hutanu D, Koesdjojo MT, Bartel CM, Remcho VT (2010) On-chip aptamer-based sandwich assay for thrombin detection employing magnetic beads and quantum dots. Anal Chem 82:5591–5597CrossRefGoogle Scholar
  38. 38.
    Xu BL, Zhao CQ, Wei WL, Ren JS, Miyoshi D, Sugimoto N, Qu XG (2012) Aptamer carbon nanodot sandwich used for fluorescent detection of protein. Analyst 137:5483–5486CrossRefGoogle Scholar
  39. 39.
    Liu YN, Liu N, Ma XH, Li XL, Ma J, Li Y, Zhou ZJ, Gao ZX (2015) Highly specific detection of thrombin using an aptamer-based suspension array and the interaction analysis via microscale thermophoresis. Analyst 140:2762–2770CrossRefGoogle Scholar
  40. 40.
    Roemhildt L, Pahlke C, Zoergiebel F, Braun H-G, Opitz J, Baraban L, Cuniberti G (2013) Patterned biochemical functionalization improves aptamer-based detection of unlabeled thrombin in a sandwich assay. ACS Appl Mater Interfaces 5:12029–12035CrossRefGoogle Scholar
  41. 41.
    Xiao SJ, Hu PP, Wu XD, Zou YL, Chen LQ, Peng L, Ling J, Zhen SJ, Zhan L, Li YF, Huang CZ (2010) Sensitive discrimination and detection of prion disease-associated isoform with a dual-aptamer strategy by developing a sandwich structure of magnetic microparticles and quantum dots. Anal Chem 82:9736–9742CrossRefGoogle Scholar
  42. 42.
    Luo Y, Liu X, Jiang TL, Liao P, Fu WL (2013) Dual-aptamer-based biosensing of toxoplasma antibody. Anal Chem 85:8354–8360CrossRefGoogle Scholar
  43. 43.
    Ruslinda AR, Penmatsa V, Ishii Y, Tajima S, Kawarada H (2012) Highly sensitive detection of platelet-derived growth factor on a functionalized diamond surface using aptamer sandwich design. Analyst 137:1692–1697CrossRefGoogle Scholar
  44. 44.
    Csordas AT, Jorgensen A, Wang J, Gruber E, Gong Q, Bagley ER, Nakamoto MA, Eisenstein M, Soh HT (2016) High-throughput discovery of aptamers for sandwich assays. Anal Chem 88:10842–10847CrossRefGoogle Scholar
  45. 45.
    Peng QW, Cao ZJ, Lau C, Kai M, Lu JZ (2011) Aptamer-barcode based immunoassay for the instantaneous derivatization chemiluminescence detection of IgE coupled to magnetic beads. Analyst 136:140–147CrossRefGoogle Scholar
  46. 46.
    Wang XQ, Ren L, Tu Q, Wang JC, Zhang YR, Li ML, Liu R, Wang JY (2011) Magnetic protein microbead-aided indirect fluoroimmunoassay for the determination of canine virus specific antibodies. Biosens Bioelectron 26:3353–3360CrossRefGoogle Scholar
  47. 47.
    Gruber HJ, Hahn CD, Kada G, Riener CK, Harms GS, Ahrer W, Dax TG, Knaus HG (2000) Anomalous fluorescence enhancement of Cy3 and Cy3.5 versus anomalous fluorescence loss of Cy5 and Cy7 upon covalent linking to IgG and noncovalent binding to avidin. Bioconjugate Chem 11:696–704CrossRefGoogle Scholar
  48. 48.
    Wang JY, Wang XQ, Ren L, Wang Q, Li L, Liu WM, Wan ZF, Yang LY, Sun P, Ren LL, Li ML, Wu H, Wang JF, Zhang L (2009) Conjugation of biomolecules with magnetic protein microspheres for the assay of early biomarkers associated with acute myocardial infarction. Anal Chem 81:6210–6217CrossRefGoogle Scholar
  49. 49.
    Wang JY, Ren L, Wang XQ, Wang Q, Wan ZF, Li L, Liu WM, Wang XM, Li ML, Tong DW, Liu AJ, Shang BB (2009) Superparamagnetic microsphere-assisted fluoroimmunoassay for rapid assessment of acute myocardial infarction. Biosens Bioelectron 24:3097–3102CrossRefGoogle Scholar
  50. 50.
    Warner MG, Grate JW, Tyler A, Ozanich RM, Miller KD, Lou J, Marks JD, Bruckner-Lea CJ (2009) Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of botulinum neurotoxin using high-affinity antibodies. Biosens Bioelectron 25:179–184CrossRefGoogle Scholar
  51. 51.
    Ziegler J, Zimmermann M, Hunziker P, Delamarche E (2008) High-performance immunoassays based on through-stencil patterned antibodies and capillary systems. Anal Chem 80:1763–1769CrossRefGoogle Scholar
  52. 52.
    Hosokawa K, Omata M, Maeda M (2007) Immunoassay on a power-free microchip with laminar flow-assisted dendritic amplification. Anal Chem 79:6000–6004CrossRefGoogle Scholar
  53. 53.
    Zhao W, Zhang WP, Zhang ZL, He RL, Lin Y, Xie M, Wang HZ, Pang DW (2012) Robust and highly sensitive fluorescence approach for point-of-care virus detection based on immunomagnetic separation. Anal Chem 84:2358–2365CrossRefGoogle Scholar
  54. 54.
    Grate JW, Warner MG, Ozanich RM Jr, Miller KD, Colburn HA, Dockendorff B, Antolick KC, Anheier NC Jr, Lind MA, Lou J, Marks JD, Bruckner-Lea CJ (2009) Renewable surface fluorescence sandwich immunoassay biosensor for rapid sensitive botulinum toxin detection in an automated fluidic format. Analyst 134:987–996CrossRefGoogle Scholar
  55. 55.
    Stern E, Vacic A, Rajan NK, Criscione JM, Park J, Ilic BR, Mooney DJ, Reed MA, Fahmy TM (2010) Label-free biomarker detection from whole blood. Nat Nanotechnol 5:138–142CrossRefGoogle Scholar
  56. 56.
    Wang JJ, Huang XY, Liu H, Dong CQ, Ren JC (2017) Fluorescence and scattering light cross correlation spectroscopy and its applications in homogeneous immunoassay. Anal Chem 89:5230–5237CrossRefGoogle Scholar
  57. 57.
    Li CY, Cao D, Qi CB, Chen HL, Wan YT, Lin Y, Zhang ZL, Pang DW, Tang HW (2017) One-step separation-free detection of carcinoembryonic antigen in whole serum: combination of two-photon excitation fluorescence and optical trapping. Biosens Bioelectron 90:146–152CrossRefGoogle Scholar
  58. 58.
    Ho SL, Xu D, Wong MS, Li HW (2016) Direct and multiplex quantification of protein biomarkers in serum samples using an immuno-magnetic platform. Chem Sci 7:2695–2700CrossRefGoogle Scholar
  59. 59.
    Yang Q, Gong X, Song T, Yang J, Zhu S, Li Y, Cui Y, Li Y, Zhang B, Chang J (2011) Quantum dot-based immunochromatography test strip for rapid, quantitative and sensitive detection of alpha fetoprotein. Biosens Bioelectron 30:145–150CrossRefGoogle Scholar
  60. 60.
    Jokerst JV, Raamanathan A, Christodoulides N, Floriano PN, Pollard AA, Simmons GW, Wong J, Gage C, Furmaga WB, Redding SW, McDevitt JT (2009) Nano-bio-chips for high performance multiplexed protein detection: determinations of cancer biomarkers in serum and saliva using quantum dot bioconjugate labels. Biosens Bioelectron 24:3622–3629CrossRefGoogle Scholar
  61. 61.
    Wu YY, Wei P, Pengpumkiat S, Schumacher EA, Remcho VT (2015) Development of a carbon dot (C-Dot)-linked immunosorbent assay for the detection of human alpha-fetoprotein. Anal Chem 87:8510–8516CrossRefGoogle Scholar
  62. 62.
    Yan JL, Estevez MC, Smith JE, Wang KM, He XX, Wang L, Tan WH (2007) Dye-doped nanoparticles for bioanalysis. Nano Today 2:44–50CrossRefGoogle Scholar
  63. 63.
    Cowles CL, Zhu XS (2011) Sensitive detection of cardiac biomarker using ZnS nanoparticles as novel signal transducers. Biosens Bioelectron 30:342–346CrossRefGoogle Scholar
  64. 64.
    Yao JJ, Han XG, Zeng S, Zhong WW (2012) Detection of femtomolar proteins by nonfluorescent ZnS nanocrystal clusters. Anal Chem 84:1645–1652CrossRefGoogle Scholar
  65. 65.
    Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li JJ, Sundaresan G, Wu AM, Gambhir SS, Weiss S (2005) Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307:538–544CrossRefGoogle Scholar
  66. 66.
    Walling MA, Novak JA, Shepard JRE (2009) Quantum dots for live cell and in vivo imaging. Int J Mol Sci 10:441–491CrossRefGoogle Scholar
  67. 67.
    Regulacio MD, Han MY (2010) Composition-tunable alloyed semiconductor nanocrystals. Acc Chem Res 43:621–630CrossRefGoogle Scholar
  68. 68.
    Chan WCW, Nie SM (1998) Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 281:2016–2018CrossRefGoogle Scholar
  69. 69.
    Cui RJ, Pan HC, Zhu JJ, Chen HY (2007) Versatile immunosensor using CdTe quantum dots as electrochemical and fluorescent labels. Anal Chem 79:8494–8501CrossRefGoogle Scholar
  70. 70.
    Tu MC, Chang YT, Kang YT, Chang HY, Chang P, Yew TR (2012) A quantum dot-based optical immunosensor for human serum albumin detection. Biosens Bioelectron 34:286–290CrossRefGoogle Scholar
  71. 71.
    Qian J, Zhang CY, Cao XD, Liu SQ (2010) Versatile immunosensor using a quantum dot coated silica nanosphere as a label for signal amplification. Anal Chem 82:6422–6429CrossRefGoogle Scholar
  72. 72.
    Mukundan H, Xie H, Price D, Kubicek-Sutherland JZ, Grace WK, Anderson AS, Martinez JS, Hartman N, Swanson BI (2010) Quantitative multiplex detection of pathogen biomarkers on multichannel waveguides. Anal Chem 82:136–144CrossRefGoogle Scholar
  73. 73.
    Gaylord BS, Heeger AJ, Bazan GC (2003) DNA hybridization detection with water-soluble conjugated polymers and chromophore-labeled single-stranded DNA. J Am Chem Soc 125:896–900CrossRefGoogle Scholar
  74. 74.
    Klingstedt T, Nilsson KPR (2011) Conjugated polymers for enhanced bioimaging. Biochim Biophys Acta Gen Subj 1810:286–296CrossRefGoogle Scholar
  75. 75.
    He F, Tang YL, Yu MH, Feng F, An LL, Sun H, Wang S, Li YL, Zhu DB, Bazan GC (2006) Quadruplex-to-duplex transition of G-rich oligonucleotides probed by cationic water-soluble conjugated polyelectrolytes. J Am Chem Soc 128:6764–6765CrossRefGoogle Scholar
  76. 76.
    Liu B, Bazan GC (2006) Optimization of the molecular orbital energies of conjugated polymers for optical amplification of fluorescent sensors. J Am Chem Soc 128:1188–1196CrossRefGoogle Scholar
  77. 77.
    Pu KY, Liu B (2009) Optimizing the cationic conjugated polymer-sensitized fluorescent signal of dye labeled oligonucleotide for biosensor applications. Biosens Bioelectron 24:1067–1073CrossRefGoogle Scholar
  78. 78.
    Wang YY, Liu B (2009) Conjugated polymer as a signal amplifier for novel silica nanoparticle-based fluoroimmunoassay. Biosens Bioelectron 24:3293–3298CrossRefGoogle Scholar
  79. 79.
    Guo LM, Hao LH, Zhao Q (2016) An aptamer assay using rolling circle amplification coupled with thrombin catalysis for protein detection. Anal Bioanal Chem 408:4715–4722CrossRefGoogle Scholar
  80. 80.
    Lee JU, Jeong JH, Lee DS, Sim SJ (2014) Signal enhancement strategy for a micro-arrayed polydiacetylene (PDA) immunosensor using enzyme-catalyzed precipitation. Biosens Bioelectron 61:314–320CrossRefGoogle Scholar
  81. 81.
    Niu SY, Qu LJ, Zhang Q, Lin JH (2012) Fluorescence detection of thrombin using autocatalytic strand displacement cycle reaction and a dual-aptamer DNA sandwich assay. Anal Biochem 421:362–367CrossRefGoogle 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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