Microchimica Acta

, 185:293 | Cite as

Non-enzymolytic adenosine barcode-mediated dual signal amplification strategy for ultrasensitive protein detection using LC-MS/MS

  • Wen Yang
  • Tengfei Li
  • Chang Shu
  • Shunli Ji
  • Lei Wang
  • Yan Wang
  • Duo Li
  • Michael Mtalimanja
  • Luning Sun
  • Li Ding
Original Paper

Abstract

A method is described for the determination of proteins with LC-MS/MS enabled by a small molecule (adenosine) barcode and based on a double-recognition sandwich structure. The coagulation protein thrombin was chosen as the model analyte. Magnetic nanoparticles were functionalized with aptamer29 (MNP/apt29) and used to capture thrombin from the samples. MNP/apt29 forms a sandwich with functionalized gold nanoparticles modified with (a) aptamer15 acting as thrombin-recognizing element and (b) a large number of adenosine as mass barcodes. The sandwich formed (MNP/apt29-thrombin-apt15/AuNP/adenosine) can ben magnetically separated from the sample. Mass barcodes are subsequently released from the sandwiched structure for further analysis by adding 11-mercaptoundecanoic acid. Adenosine is then detected by LC-MS/MS as it reflects the level of thrombin with impressively amplified signal. Numerous adenosines introduced into the sandwich proportional to the target concentration further amplify the signal. Under optimized conditions, the response is linearly proportional to the thrombin concentration in the range of 0.02 nM to 10 nM, with a detection limit of 9 fM. The application of this method to the determination of thrombin in spiked plasma samples gave recoveries that ranged from 92.3% to 104.7%.

Graphical abstract

Schematic representation of a method for the determination of thrombin with LC-MS/MS. The method is based on a double-recognition sandwiched structure. With LC-MS/MS, mass barcodes (adenosine) are detected to quantify thrombin, which amplifies the detection signal impressively.

Keywords

Signal transduction Nanocomposites Mass barcodes Aptamer Proteins 

Notes

Acknowledgments

We are grateful for the financial support of the National Natural Science Foundation of China (No.81573387, No.81703472, No.81603072 and No.81560695). Also thanks for the support from the Postgraduate Research & Practice Innovation Program of Jiangsu Province (No. KYCX17_0683).

Compliance with ethical standards

The author(s) declare that they have no competing interests.

Ethical approval

Institutional Review Board approval was not required because the study is not on animals.

Informed consent

Written informed consent was not required for this study because the study is not on human subjects.

Supplementary material

604_2018_2832_MOESM1_ESM.doc (400 kb)
ESM 1 (DOC 399 kb)

References

  1. 1.
    Khalil DN, Smith EL, Brentjens RJ, Wolchok JD (2016) The future of cancer treatment: immunomodulation, CARs and combination immunotherapy. Nat Rev Clin Oncol 13(5):273–290.  https://doi.org/10.1038/nrclinonc.2016.25 CrossRefGoogle Scholar
  2. 2.
    Benjamin Leader QJB, Golan DE (2008) Protein therapeutics: a summary and pharmacological classification. Nat Rev Drug Discov 7:21–39CrossRefGoogle Scholar
  3. 3.
    Lequin RM (2005) Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA). Clin Chem 51(12):2415–2418.  https://doi.org/10.1373/clinchem.2005.051532 CrossRefGoogle Scholar
  4. 4.
    Giljohann DA, Mirkin CA (2009) Drivers of biodiagnostic development. Nature 462(7272):461–464.  https://doi.org/10.1038/nature08605 CrossRefGoogle Scholar
  5. 5.
    Yunlei Xianyu ZW, Jiang X (2014) A plasmonic nanosensor for immunoassay via enzyme-triggered click chemistry. ACS Nano 8(12):12741–12747CrossRefGoogle Scholar
  6. 6.
    Ma X, Chen Z, Kannan P, Lin Z, Qiu B, Guo L (2016) Gold nanorods as colorful chromogenic substrates for semiquantitative detection of nucleic acids, proteins, and small molecules with the naked eye. Anal Chem 88(6):3227–3234.  https://doi.org/10.1021/acs.analchem.5b04621 CrossRefGoogle Scholar
  7. 7.
    Zhan L, Guo SZ, Song F, Gong Y, Xu F, Boulware DR, McAlpine MC, Chan WCW, Bischof JC (2017) The role of nanoparticle Design in Determining Analytical Performance of lateral flow immunoassays. Nano Lett 17(12):7207–7212.  https://doi.org/10.1021/acs.nanolett.7b02302 CrossRefGoogle Scholar
  8. 8.
    Yu CC, Kuo YY, Liang CF, Chien WT, Wu HT, Chang TC, Jan FD, Lin CC (2012) Site-specific immobilization of enzymes on magnetic nanoparticles and their use in organic synthesis. Bioconjug Chem 23(4):714–724.  https://doi.org/10.1021/bc200396r CrossRefGoogle Scholar
  9. 9.
    Zhou J, Gan N, Li T, Zhou H, Li X, Cao Y, Wang L, Sang W, Hu F (2013) Ultratrace detection of C-reactive protein by a piezoelectric immunosensor based on Fe3O4@SiO2 magnetic capture nanoprobes and HRP-antibody co-immobilized nano gold as signal tags. Sensors Actuators B Chem 178:494–500.  https://doi.org/10.1016/j.snb.2013.01.013 CrossRefGoogle Scholar
  10. 10.
    Zhao Q, Li XF, Le XC (2011) Aptamer capturing of enzymes on magnetic beads to enhance assay specificity and sensitivity. Anal Chem 83(24):9234–9236.  https://doi.org/10.1021/ac203063z CrossRefGoogle Scholar
  11. 11.
    Piella J, Bastús NG, Puntes V (2016) Size-controlled synthesis of Sub-10-nanometer citrate-stabilized gold nanoparticles and related optical properties. Chem Mater 28(4):1066–1075.  https://doi.org/10.1021/acs.chemmater.5b04406 CrossRefGoogle Scholar
  12. 12.
    Schuetze B, Mayer C, Loza K, Gocyla M, Heggen M, Epple M (2016) Conjugation of thiol-terminated molecules to ultrasmall 2 nm-gold nanoparticles leads to remarkably complex 1H-NMR spectra. J Mater Chem B 4(12):2179–2189.  https://doi.org/10.1039/c5tb02443a CrossRefGoogle Scholar
  13. 13.
    Azubel M, Kornberg RD (2016) Synthesis of water-soluble, thiolate-protected gold nanoparticles uniform in size. Nano Lett 16(5):3348–3351.  https://doi.org/10.1021/acs.nanolett.6b00981 CrossRefGoogle Scholar
  14. 14.
    Zhou J, Rossi J (2017) Aptamers as targeted therapeutics: current potential and challenges. Nat Rev Drug Discov 16(3):181–202.  https://doi.org/10.1038/nrd.2016.199 CrossRefGoogle Scholar
  15. 15.
    van den Kieboom CH, van der Beek SL, Mészáros T, Gyurcsányi RE, Ferwerda G, de Jonge MI (2015) Aptasensors for viral diagnostics. TrAC Trends Anal Chem 74:58–67.  https://doi.org/10.1016/j.trac.2015.05.012 CrossRefGoogle Scholar
  16. 16.
    Liu Z, Liu H, Wang L, Su X (2016) A label-free fluorescence biosensor for highly sensitive detection of lectin based on carboxymethyl chitosan-quantum dots and gold nanoparticles. Anal Chim Acta 932:88–97.  https://doi.org/10.1016/j.aca.2016.05.025 CrossRefGoogle Scholar
  17. 17.
    Wang L, Yang W, Li T, Li D, Cui Z, Wang Y, Ji S, Song Q, Shu C, Ding L (2017) Colorimetric determination of thrombin by exploiting a triple enzyme-mimetic activity and dual-aptamer strategy. Microchim Acta 184(9):3145–3151.  https://doi.org/10.1007/s00604-017-2327-8 CrossRefGoogle Scholar
  18. 18.
    Meirinho SG, Dias LG, Peres AM, Rodrigues LR (2017) Electrochemical aptasensor for human osteopontin detection using a DNA aptamer selected by SELEX. Anal Chim Acta 987:25–37.  https://doi.org/10.1016/j.aca.2017.07.071 CrossRefGoogle Scholar
  19. 19.
    Cheng Z, Choi N, Wang R, Lee S, Moon KC, Yoon SY, Chen L, Choo J (2017) Simultaneous detection of dual prostate specific antigens using surface-enhanced Raman scattering-based immunoassay for accurate diagnosis of prostate cancer. ACS Nano 11(5):4926–4933.  https://doi.org/10.1021/acsnano.7b01536 CrossRefGoogle Scholar
  20. 20.
    El-Said WA, Fouad DM, El-Safty SA (2016) Ultrasensitive label-free detection of cardiac biomarker myoglobin based on surface-enhanced Raman spectroscopy. Sensors Actuators B Chem 228:401–409.  https://doi.org/10.1016/j.snb.2016.01.041 CrossRefGoogle Scholar
  21. 21.
    Jazayeri MH, Amani H, Pourfatollah AA, Avan A, Ferns GA, Pazoki-Toroudi H (2016) Enhanced detection sensitivity of prostate-specific antigen via PSA-conjugated gold nanoparticles based on localized surface plasmon resonance: GNP-coated anti-PSA/LSPR as a novel approach for the identification of prostate anomalies. Cancer Gene Ther 23(10):365–369.  https://doi.org/10.1038/cgt.2016.42 CrossRefGoogle Scholar
  22. 22.
    Joanna Zheng JM, Yongxin Zhu BX, Olah T (2014) Application and challenges in using LC–MS assays for absolute quantitative analysis of therapeutic proteins in drug discovery. Bioanalysis 6(6):21Google Scholar
  23. 23.
    Li H, Ortiz R, Tran LT, Salimi-Moosavi H, Malella J, James CA, Lee JW (2013) Simultaneous analysis of multiple monoclonal antibody biotherapeutics by LC-MS/MS method in rat plasma following cassette-dosing. AAPS J 15(2):337–346.  https://doi.org/10.1208/s12248-012-9435-5 CrossRefGoogle Scholar
  24. 24.
    An B, Zhang M, Johnson RW, Qu J (2015) Surfactant-aided precipitation/on-pellet-digestion (SOD) procedure provides robust and rapid sample preparation for reproducible, accurate and sensitive LC/MS quantification of therapeutic protein in plasma and tissues. Anal Chem 87(7):4023–4029.  https://doi.org/10.1021/acs.analchem.5b00350 CrossRefGoogle Scholar
  25. 25.
    Liu J, Lu Y (2006) Preparation of aptamer-linked gold nanoparticle purple aggregates for colorimetric sensing of analytes. Nat Protoc 1(1):246–252.  https://doi.org/10.1038/nprot.2006.38 CrossRefGoogle Scholar
  26. 26.
    Sheng H, Ye BC (2009) Different strategies of covalent attachment of oligonucleotide probe onto glass beads and the hybridization properties. Appl Biochem Biotechnol 152(1):54–65.  https://doi.org/10.1007/s12010-008-8245-9 CrossRefGoogle Scholar
  27. 27.
    Jwa-Min Nam CST, Mirkint CA (2003) Nanoparticle-based bio-bar codes for the ultrasensitive detection of proteins. Science 301(5641):3Google Scholar
  28. 28.
    Bernd Giese DM (2002) Surface-enhanced Raman spectroscopic and density functional theory study of adenine adsorption to silver surfaces. J Phys Chem B 106:12CrossRefGoogle Scholar
  29. 29.
    Kundu J, ON BGJ, Zhang D, Lal S, Barhoumi A, Scuseria GE, Halas NJ (2009) Adenine− and adenosine monophosphate (AMP)−gold binding interactions studied by surface-enhanced Raman and infrared spectroscopies. J Phys Chem C 113:8Google Scholar
  30. 30.
    Siriwardana K, Gadogbe M, Ansar SM, Vasquez ES, Collier WE, Zou S, Walters KB, Zhang D (2014) Ligand adsorption and exchange on Pegylated gold nanoparticles. J Phys Chem C 118(20):11111–11119.  https://doi.org/10.1021/jp501391x CrossRefGoogle Scholar
  31. 31.
    Zhuo B, Li Y, Huang X, Lin Y, Chen Y, Gao W (2015) An electrochemiluminescence aptasensing platform based on ferrocene-graphene nanosheets for simple and rapid detection of thrombin. Sensors Actuators B Chem 208:518–524.  https://doi.org/10.1016/j.snb.2014.11.064 CrossRefGoogle Scholar
  32. 32.
    Bai Y, Feng F, Zhao L, Wang C, Wang H, Tian M, Qin J, Duan Y, He X (2013) Aptamer/thrombin/aptamer-AuNPs sandwich enhanced surface plasmon resonance sensor for the detection of subnanomolar thrombin. Biosens Bioelectron 47:265–270.  https://doi.org/10.1016/j.bios.2013.02.004 CrossRefGoogle Scholar
  33. 33.
    Liu X, Hua X, Fan Q, Chao J, Su S, Huang YQ, Wang L, Huang W (2015) Thioflavin T as an efficient G-quadruplex inducer for the highly sensitive detection of thrombin using a new foster resonance energy transfer system. ACS Appl Mater Interfaces 7(30):16458–16465.  https://doi.org/10.1021/acsami.5b03662 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Pharmaceutical AnalysisChina Pharmaceutical UniversityNanjingChina
  2. 2.Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of EducationChina Pharmaceutical UniversityNanjingChina
  3. 3.College of PharmacyAnhui University of Chinese MedicineHefeiChina
  4. 4.College of Pharmacy and ChemistryDali UniversityDaliChina
  5. 5.Research Division of Clinical PharmacologyFirst Affiliated Hospital with Nanjing Medical UniversityNanjingChina

Personalised recommendations