Biosensors Based on Supersandwich Assays

  • Xiaojin ZhangEmail author
  • Fan Xia


Only one signal probe is usually bound to the target in traditional sandwich assays, which limits the detection sensitivity. To overcome this limitation, supersandwich assays amplifying the signal through integration of multiple signal probes together have been developed in recent years. In this chapter, we highlight biosensors based on supersandwich assays for the detection of proteins, nucleic acids, small molecules, ions, and cells by a series of efforts reported in the past decade. The detection technologies employed in design of biosensors based on supersandwich assays contain electrochemical assay, electrochemiluminescence assay, fluorescence assay, and surface plasmon resonance assay.


Supersandwich assays Multiple signal probes Electrochemical assay Electrochemiluminescence assay Fluorescence assay Surface plasmon resonance assay 


  1. 1.
    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
  2. 2.
    Xia F, White RJ, Zuo XL, Patterson A, Xiao Y, Kang D, Gong X, Plaxco KW, Heeger AJ (2010) An electrochemical supersandwich assay for sensitive and selective DNA detection in complex matrices. J Am Chem Soc 132:14346–14348CrossRefGoogle Scholar
  3. 3.
    Liu NN, Huang FJ, Lou XD, Xia F (2017) DNA hybridization chain reaction and DNA supersandwich self-assembly for ultrasensitive detection. Sci China-Chem 60:311–318CrossRefGoogle Scholar
  4. 4.
    Wang GF, Huang H, Wang BJ, Zhang XJ, Wang L (2012) A supersandwich multienzyme-DNA label based electrochemical immunosensor. Chem Commun 48:720–722CrossRefGoogle Scholar
  5. 5.
    Wang GF, He XP, Wang L, Zhang XJ (2013) A folate receptor electrochemical sensor based on terminal protection and supersandwich DNAzyme amplification. Biosens Bioelectron 42:337–341CrossRefGoogle Scholar
  6. 6.
    Wang GF, He XP, Zhu YH, Chen L, Wang L, Zhang XJ (2013) G-quadruplex-linked supersandwich DNA structure for electrochemical amplified detection of thrombin. Electroanalysis 25:1960–1966CrossRefGoogle Scholar
  7. 7.
    Wang Q, Liu W, Xing YQ, Yang XH, Wang KM, Jiang R, Wang P, Zhao Q (2014) Screening of DNA aptamers against myoglobin using a positive and negative selection units integrated microfluidic chip and its biosensing application. Anal Chem 86:6572–6579CrossRefGoogle Scholar
  8. 8.
    Liu HP, Chen Y, He Y, Ribbe AE, Mao CD (2006) Approaching the limit: can one DNA oligonucleotide assemble into large nanostructures? Angew Chem Int Ed 45:1942–1945CrossRefGoogle Scholar
  9. 9.
    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
  10. 10.
    Chen L, Sha L, Qiu YW, Wang GF, Jiang H, Zhang XJ (2015) An amplified electrochemical aptasensor based on hybridization chain reactions and catalysis of silver nanoclusters. Nanoscale 7:3300–3308CrossRefGoogle Scholar
  11. 11.
    Zhou FY, Yao Y, Luo JJ, Zhang X, Zhang Y, Yin DY, Gao FL, Wang P (2017) Proximity hybridization-regulated catalytic DNA hairpin assembly for electrochemical immunoassay based on in situ DNA template-synthesized Pd nanoparticles. Anal Chim Acta 969:8–17CrossRefGoogle Scholar
  12. 12.
    Smith ZD, Meissner A (2013) DNA methylation: roles in mammalian development. Nat Rev Genet 14:204–220CrossRefGoogle Scholar
  13. 13.
    Klutstein M, Nejman D, Greenfield R, Cedar H (2016) DNA methylation in cancer and aging. Cancer Res 76:3446–3450CrossRefGoogle Scholar
  14. 14.
    Edwards JR, Yarychkivska O, Boulard M, Bestor TH (2017) DNA methylation and DNA methyltransferases. Epigenetics Chromatin 10:23CrossRefGoogle Scholar
  15. 15.
    Duan XR, Liu LB, Feng FD, Wang S (2010) Cationic conjugated polymers for optical detection of DNA methylation, lesions, and single nucleotide polymorphisms. Acc Chem Res 43:260–270CrossRefGoogle Scholar
  16. 16.
    Flusberg BA, Webster DR, Lee JH, Travers KJ, Olivares EC, Clark TA, Korlach J, Turner SW (2010) Direct detection of DNA methylation during single-molecule, real-time sequencing. Nat Methods 7:461–465CrossRefGoogle Scholar
  17. 17.
    Li Y, Luo XE, Yan Z, Zheng JB, Qi HL (2013) A label-free supersandwich electrogenerated chemiluminescence method for the detection of DNA methylation and assay of the methyltransferase activity. Chem Commun 49:3869–3871CrossRefGoogle Scholar
  18. 18.
    Sun HP, Ma SX, Li Y, Qi HL (2017) Electrogenerated chemiluminescence biosensing method for the detection of DNA demethylase activity: combining MoS2 nanocomposite with DNA supersandwich. Sens Actuator B-Chem 244:885–890CrossRefGoogle Scholar
  19. 19.
    Jiang BY, Yang ML, Yang CY, Xiang Y, Yuan R (2017) Methylation-induced inactivation of restriction enzyme for amplified and signal-on electrochemiluminescence detection of methyltransferase activity. Sens Actuator B-Chem 247:573–579CrossRefGoogle Scholar
  20. 20.
    Lei R, Wang XY, Zhu SF, Li N (2011) A novel electrochemiluminescence glucose biosensor based on alcohol-free mesoporous molecular sieve silica modified electrode. Sens Actuator B-Chem 158:124–129CrossRefGoogle Scholar
  21. 21.
    Gui GF, Zhuo Y, Chai YQ, Liao N, Zhao M, Han J, Zhu Q, Yuan R, Xiang Y (2013) Supersandwich-type electrochemiluminescenct aptasensor based on Ru(phen)32+ functionalized hollow gold nanoparticles as signal-amplifying tags. Biosens Bioelectron 47:524–529CrossRefGoogle Scholar
  22. 22.
    He Y, Chai YQ, Yuan R, Wang HJ, Bai LJ, Cao YL, Yuan YL (2013) An ultrasensitive electrochemiluminescence immunoassay based on supersandwich DNA structure amplification with histidine as a co-reactant. Biosens Bioelectron 50:294–299CrossRefGoogle Scholar
  23. 23.
    He Y, Chai YQ, Yuan R, Wang HJ, Bai LJ, Liao N (2014) A supersandwich electrochemiluminescence immunosensor based on mimic-intramolecular interaction for sensitive detection of proteins. Analyst 139:5209–5214CrossRefGoogle Scholar
  24. 24.
    Zhou LY, Zhang XY, Wang GL, Jiao XX, Luo HQ, Li NB (2012) A simple and label-free electrochemical biosensor for DNA detection based on the super-sandwich assay. Analyst 137:5071–5075CrossRefGoogle Scholar
  25. 25.
    Chen Y, Wang Q, Xu J, Xiang Y, Yuan R, Chai YQ (2013) A new hybrid signal amplification strategy for ultrasensitive electrochemical detection of DNA based on enzyme-assisted target recycling and DNA supersandwich assemblies. Chem Commun 49:2052–2054CrossRefGoogle Scholar
  26. 26.
    Wang J, Shi AQ, Fang X, Han XW, Zhang YZ (2014) Ultrasensitive electrochemical supersandwich DNA biosensor using a glassy carbon electrode modified with gold particle-decorated sheets of graphene oxide. Microchim Acta 181:935–940CrossRefGoogle Scholar
  27. 27.
    Wang J, Shi AQ, Fang X, Han XW, Zhang YZ (2015) An ultrasensitive supersandwich electrochemical DNA biosensor based on gold nanoparticles decorated reduced graphene oxide. Anal Biochem 469:71–75CrossRefGoogle Scholar
  28. 28.
    Wei BM, Liu NN, Zhang JT, Ou XW, Duan RX, Yang ZK, Lou XD, Xia F (2015) Regulation of DNA self-assembly and DNA hybridization by chiral molecules with corresponding biosensor applications. Anal Chem 87:2058–2062CrossRefGoogle Scholar
  29. 29.
    Ren W, Zhou LY, Zhang Y, Li NB, Luo HQ (2016) A reusable and label-free supersandwich biosensor for sensitive DNA detection by immobilizing target-triggered DNA concatamers on ternary self-assembled monolayer. Sens Actuator B-Chem 223:24–29CrossRefGoogle Scholar
  30. 30.
    Wei BM, Zhang TC, Ou XW, Li XC, Lou XD, Xia F (2016) Stereochemistry-guided DNA probe for single nucleotide polymorphisms analysis. ACS Appl Mater Interfaces 8:15911–15916CrossRefGoogle Scholar
  31. 31.
    Zhang H, Wang Q, Yang XH, Wang KM, Li Q, Li ZP, Gao L, Nie WY, Zheng Y (2017) An isothermal electrochemical biosensor for the sensitive detection of microRNA based on a catalytic hairpin assembly and supersandwich amplification. Analyst 142:389–396CrossRefGoogle Scholar
  32. 32.
    Liu NN, Jiang YN, Zhou YH, Xia F, Guo W, Jiang L (2013) Two-way nanopore sensing of sequence-specific oligonucleotides and small-molecule targets in complex matrices using integrated DNA supersandwich structures. Angew Chem Int Ed 52:2007–2011CrossRefGoogle Scholar
  33. 33.
    Ruan SP, Li ZJ, Qi HL, Gao Q, Zhang CX (2014) Label-free supersandwich electrogenerated chemiluminescence biosensor for the determination of the HIV gene. Microchim Acta 181:1293–1300CrossRefGoogle Scholar
  34. 34.
    Yu JH, Choi S, Dickson RM (2009) Shuttle-based fluorogenic silver-cluster biolabels. Angew Chem Int Ed 48:318–320CrossRefGoogle Scholar
  35. 35.
    Wang GF, Zhu YH, Chen L, Wang L, Zhang XJ (2014) Target-induced quenching for highly sensitive detection of nucleic acids based on label-free luminescent supersandwich DNA/silver nanoclusters. Analyst 139:165–169CrossRefGoogle Scholar
  36. 36.
    Huang J, Wang H, Yang XH, Yang YJ, Quan K, Ying L, Xie NL, Ou M, Wang KM (2016) A supersandwich fluorescence in situ hybridization strategy for highly sensitive and selective mRNA imaging in tumor cells. Chem Commun 52:370–373CrossRefGoogle Scholar
  37. 37.
    Homola J (2008) Surface plasmon resonance sensors for detection of chemical and biological species. Chem Rev 108:462–493CrossRefGoogle Scholar
  38. 38.
    Ding XJ, Yan YR, Li SQ, Zhang Y, Cheng W, Cheng Q, Ding SJ (2015) Surface plasmon resonance biosensor for highly sensitive detection of microRNA based on DNA super-sandwich assemblies and streptavidin signal amplification. Anal Chim Acta 874:59–65CrossRefGoogle Scholar
  39. 39.
    Wang Q, Liu RJ, Yang XH, Wang KM, Zhu JQ, He LL, Li Q (2016) Surface plasmon resonance biosensor for enzyme-free amplified microRNA detection based on gold nanoparticles and DNA supersandwich. Sens Actuator B-Chem 223:613–620CrossRefGoogle Scholar
  40. 40.
    Liu RJ, Wang Q, Li Q, Yang XH, Wang KM, Nie WY (2017) Surface plasmon resonance biosensor for sensitive detection of microRNA and cancer cell using multiple signal amplification strategy. Biosens Bioelectron 87:433–438CrossRefGoogle Scholar
  41. 41.
    Patel A, Malinovska L, Saha S, Wang J, Alberti S, Krishnan Y, Hyman AA (2017) ATP as a biological hydrotrope. Science 356:753–756CrossRefGoogle Scholar
  42. 42.
    Ma CB, Chen HC, Han R, He HL, Zeng WM (2012) Fluorescence detection of adenosine triphosphate using smart probe. Anal Biochem 429:8–10CrossRefGoogle Scholar
  43. 43.
    Huo Y, Qi L, Lv XJ, Lai T, Zhang J, Zhang ZQ (2016) A sensitive aptasensor for colorimetric detection of adenosine triphosphate based on the protective effect of ATP-aptamer complexes on unmodified gold nanoparticles. Biosens Bioelectron 78:315–320CrossRefGoogle Scholar
  44. 44.
    Wei BM, Zhang JT, Wang HB, Xia F (2016) A new electrochemical aptasensor based on a dual-signaling strategy and supersandwich assay. Analyst 141:4313–4318CrossRefGoogle Scholar
  45. 45.
    Jiang YN, Liu NN, Guo W, Xia F, Jiang L (2012) Highly-efficient gating of solid-state nanochannels by DNA supersandwich structure containing ATP aptamers: a nanofluidic implication logic device. J Am Chem Soc 134:15395–15401CrossRefGoogle Scholar
  46. 46.
    Yang XH, Zhu JQ, Wang Q, Wang KM, Yang LJ, Zhu HZ (2012) A label-free and sensitive supersandwich electrochemical biosensor for small molecule detection based on target-induced aptamer displacement. Anal Methods 4:2221–2223CrossRefGoogle Scholar
  47. 47.
    Wang GF, He XP, Chen L, Zhu YH, Zhang XJ, Wang L (2013) Conformational switch for cisplatin with hemin/G-quadruplex DNAzyme supersandwich structure. Biosens Bioelectron 50:210–216CrossRefGoogle Scholar
  48. 48.
    Ono A, Togashi H (2004) Highly selective oligonucleotide-based sensor for mercury(II) in aqueous solutions. Angew Chem Int Ed 43:4300–4302CrossRefGoogle Scholar
  49. 49.
    Wang GF, He XP, Wang BJ, Zhang XJ, Wang L (2012) Electrochemical amplified detection of Hg2+ based on the supersandwich DNA structure. Analyst 137:2036–2038CrossRefGoogle Scholar
  50. 50.
    AshaRani PV, Mun GLK, Hande MP, Valiyaveettil S (2009) Cytotoxicity and genotoxicity of silver nanoparticles in human cells. ACS Nano 3:279–290CrossRefGoogle Scholar
  51. 51.
    Xu G, Wang GF, He XP, Zhu YH, Chen L, Zhang XJ (2013) An ultrasensitive electrochemical method for detection of Ag+ based on cyclic amplification of exonuclease III activity on cytosine-Ag+-cytosine. Analyst 138:6900–6906CrossRefGoogle Scholar
  52. 52.
    Zhang YL, Li HY, Chen M, Fang X, Pang PF, Wang HB, Wu Z, Yang WR (2017) Ultrasensitive electrochemical biosensor for silver ion based on magnetic nanoparticles labeling with hybridization chain reaction amplification strategy. Sens Actuator B-Chem 249:431–438CrossRefGoogle Scholar
  53. 53.
    Liu NN, Hou RZ, Gao PC, Lou XD, Xia F (2016) Sensitive Zn2+ sensor based on biofunctionalized nanopores via combination of DNAzyme and DNA supersandwich structures. Analyst 141:3626–3629CrossRefGoogle Scholar
  54. 54.
    Chen Y, Yang ML, Xiang Y, Yuan R, Chai YQ (2014) Binding-induced autonomous disassembly of aptamer-DNAzyme supersandwich nanostructures for sensitive electrochemiluminescence turn-on detection of ochratoxin A. Nanoscale 6:1099–1104CrossRefGoogle Scholar
  55. 55.
    Yuan T, Liu ZY, Hu LZ, Zhang L, Xu GB (2011) Label-free supersandwich electrochemiluminescence assay for detection of sub-nanomolar Hg2+. Chem Commun 47:11951–11953CrossRefGoogle Scholar
  56. 56.
    Lin CS, Chen YY, Cai ZX, Zhu Z, Jiang YQ, Yang CJ, Chen X (2015) A label-free fluorescence strategy for sensitive detection of ATP based on the ligation-triggered super-sandwich. Biosens Bioelectron 63:562–565CrossRefGoogle Scholar
  57. 57.
    Yuan T, Hu LZ, Liu ZY, Qi WJ, Zhu SY, Aziz ur R, Xu GB (2013) A label-free and signal-on supersandwich fluorescent platform for Hg2+ sensing. Anal Chim Acta 793:86–89CrossRefGoogle Scholar
  58. 58.
    Galanzha EI, Shashkov EV, Kelly T, Kim JW, Yang LL, Zharov VP (2009) In vivo magnetic enrichment and multiplex photoacoustic detection of circulating tumour cells. Nat Nanotechnol 4:855–860CrossRefGoogle Scholar
  59. 59.
    Liu HY, Xu SM, He ZM, Deng AP, Zhu JJ (2013) Supersandwich cytosensor for selective and ultrasensitive detection of cancer cells using aptamer-DNA concatamer-quantum dots probes. Anal Chem 85:3385–3392CrossRefGoogle Scholar
  60. 60.
    Lu CY, Xu JJ, Wang ZH, Chen HY (2015) A novel signal-amplified electrochemical aptasensor based on supersandwich G-quadruplex DNAzyme for highly sensitive cancer cell detection. Electrochem Commun 52:49–52CrossRefGoogle Scholar
  61. 61.
    Li N, Xiao TY, Zhang ZT, He RX, Wen D, Cao YP, Zhang WY, Chen Y (2015) A 3D graphene oxide microchip and a Au-enwrapped silica nanocomposite-based supersandwich cytosensor toward capture and analysis of circulating tumor cells. Nanoscale 7:16354–16360CrossRefGoogle Scholar

Copyright information

© 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 Chemistryand Materia Medica, School of Chemistry and Chemical EngineeringHuazhong University of Science and TechnologyWuhanPeople’s Republic of China

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