Microchimica Acta

, 186:460 | Cite as

Enzymatic determination of D-alanine using a cationic poly(fluorenylenephenylene) as the fluorescent probe and MnO2 nanosheets as quenchers

  • Ye Lu
  • Yong Liu
  • Chenchen Wang
  • Shuangshuang Wu
  • Kai Zhou
  • Wei WeiEmail author
Original Paper


A sensitive fluorometric assay is described here for sensitive determination of D-alanine (D-Ala). The method is based on the use of poly[9,9-bis(6-N,N,N-trimethylammonium)hexyl]fluorenylene phenylene (PFP) and manganese dioxide (MnO2) nanosheets. The blue fluorescence of PFP, peaking at 422 nm, is absorbed by the MnO2 nanosheets due to an inner filter effect (IFE). In the presence of D-Ala and D-α-amino acid oxidase (D-AAO), the enzymatic oxidation leads to the production of H2O2, triggering the decomposition of MnO2 nanosheets and the recovery of the fluorescence of PFP. Under the optimum conditions, the nanosensor has a wide linear range from 1.0 nM to 1.0 mM with a detection limit of 0.35 nM. This is about 10–100 times lower than most previously reported methods. The recovery experiment was performed with spiked serum and gave accuracy rates from 97.2 to 109%. The standard deviation is from 2.3% to 7.2%.

Graphical abstract

Schematic presentation of a sensitive “off-on” nanosensor for fluorimetric D-alanine detection.


Cationic conjugated polymer Nanosensor Amino acid oxidase Gastric cancer Inner filter effect 



This work was supported by the National Natural Science Foundation of China (Nos. 21775019, 21635004). Fundamental Research Funds for the Central Universities and A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (Nos. 2242018K3DN04), The Open Project of The Key Laboratory of Modern Toxicology of Ministry of Education, Nanjing Medical University (NMUMT201804).

Compliance with ethical standards

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


  1. 1.
    Siegel R, Jiemin Ma PD, Zou Z, Dvm AJ (2014) Cancer statistics. CA Cancer J Clin 64(1):9–29CrossRefGoogle Scholar
  2. 2.
    Torre LA, Bray F, Siegel RL, Ferlay J, Jdft L-T Jemal A(2015) Global cancer statistics. CA Cancer J Clin 65(2):87–108PubMedCrossRefGoogle Scholar
  3. 3.
    Jung DH, Lee YC, Kim JH, Park JJ, Youn YH, Park H (2016) Additive treatment improves survival in elderly patients after non-curative endoscopic resection for early gastric cancer. Surg Endosc 31(3):1–7Google Scholar
  4. 4.
    Suzuki H, Oda I, Abe S, Yoshinaga S, Saito Y (2016) High rate of 5-year survival among patients with early gastric cancer undergoing curative endoscopic submucosal dissection. Gastric Cancer 19(1):198–205PubMedCrossRefGoogle Scholar
  5. 5.
    Kawazoe T, Tsuge HM, Fukui K (2010) Crystal structure of human d-amino acid oxidase: context-dependent variability of the backbone conformation of the VAAGL hydrophobic stretch located at the si-face of the flavin ring. Protein Sci 15(12):2708–2717CrossRefGoogle Scholar
  6. 6.
    Nagata Y, Sato T, Enomoto N, Ishii Y, Sasaki K, Yamada T (2007) High concentrations of d-amino acids in human gastric juice. Amino Acids 32(1):137–140PubMedCrossRefGoogle Scholar
  7. 7.
    Pundir CS, Lata S, Narwal V (2018) Biosensors for determination of d and l- amino acids: a review. Biosens Bioelectron 117:373–384PubMedCrossRefGoogle Scholar
  8. 8.
    Zhang ZK, Liu Y, Liu PF, Jiang XY, Lou D, Yang DY (2017) Non-invasive detection of gastric cancer relevant d-amino acids with luminescent DNA/silver nanoclusters. Nanoscale 9(48):19367–19373PubMedCrossRefGoogle Scholar
  9. 9.
    Inaba Y, Mizukami K, Hamada-Sato N, Kobayashi T, Imada C, Watanabe E (2003) Development of a d-alanine sensor for the monitoring of a fermentation using the improved selectivity by the combination of d-amino acid oxidase and pyruvate oxidase. Biosens Bioelectron 19(5):423–431PubMedCrossRefGoogle Scholar
  10. 10.
    Khoronenkova SV, Tishkov VI (2008) D-amino acid oxidase: physiological role and applications. Biochemistry-Moscow 73(13):1511–1518PubMedCrossRefGoogle Scholar
  11. 11.
    Sun J, Du K, Song XQ, Ma JJ, Ji PJ, Feng W (2015) Specific immobilization of d-amino acid oxidase on hematin-functionalized support mimicking multi-enzyme catalysis. Green Chem 17(8):4465–4472CrossRefGoogle Scholar
  12. 12.
    Sarkar P, Tothill IE, Setford SJ, Turner APF (1999) Screen-printed amperometric biosensors for the rapid measurement of l- and d-amino acids. Analyst 124(6):865–870PubMedCrossRefGoogle Scholar
  13. 13.
    Mora MF, Giacomelli CE, Garcia CD, Garcia CD (2009) Interaction of D-amino acid oxidase with carbon nanotubes: implications in the design of biosensors. Anal Chem 81(3):1016–1022PubMedPubMedCentralCrossRefGoogle Scholar
  14. 14.
    Molla G, Sacchi S, Bernasconi M, Pilone MS, Fukui K, Pollegioni L (2006) Characterization of human d-amino acid oxidase. FEBS Lett 580(9):2358–2364PubMedCrossRefGoogle Scholar
  15. 15.
    Lin X, Zhu S, Xia Q, Ma J, Fu YZ (2017) An ultrasensitive electrochemiluminescent d-alanine biosensor based on the synergetic catalysis of a hemin-functionalized composite and gold–platinum nanowires. Anal Methods 10(1):84–90CrossRefGoogle Scholar
  16. 16.
    Lata S, Batra B, Karwasra N, Pundir CS (2012) An amperometric H2O2 biosensor based on cytochrome cimmobilized onto nickel oxide nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline modified gold electrode. Process Biochem 47(6):992–998CrossRefGoogle Scholar
  17. 17.
    Lata S, Batra B, Kumar P, Pundir CS (2013) Construction of an amperometric d-amino acid biosensor based on d-amino acid oxidase/carboxylated mutliwalled carbon nanotube/copper nanoparticles/polyalinine modified gold electrode. Anal Biochem 437(1):1–9PubMedCrossRefGoogle Scholar
  18. 18.
    Hamase K, Morikawa A, Ohgusu T, Lindner W, Zaitsu K (2007) Comprehensive analysis of branched aliphatic d-amino acids in mammals using an integrated multi-loop two-dimensional column-switching high-performance liquid chromatographic system combining reversed-phase and enantioselective columns. J Chromatogr A 1143(1–2):105–111PubMedCrossRefGoogle Scholar
  19. 19.
    Rubio-Barroso S, Santos-Delgado MJ, Martín-Olivar C, Polo-Diez LM (2006) Indirect chiral HPLC determination and fluorimetric detection of d-amino acids in milk and oyster samples. J Dairy Sci 89(1):82–89PubMedCrossRefGoogle Scholar
  20. 20.
    Takano Y, Chikaraishi Y, Ogawa NO, Kitazato H, Ohkouchi N (2009) Compound-specific nitrogen isotope analysis of d-alanine, l-alanine, and valine: application of diastereomer separation to delta n-15 and microbial peptidoglycan studies. Anal Chem 81(1):394–399PubMedCrossRefGoogle Scholar
  21. 21.
    Carlavilla D, Moreno-Arribas MV, Fanali S, Cifuentes A (2006) Chiral MEKC-LIF of amino acids in foods: analysis of vinegars. Electrophoresis 27(13):2551–2557PubMedCrossRefGoogle Scholar
  22. 22.
    Inaba Y, Hamada-Sato N, Kobayashi T, Imada C, Watanabe E (2003) Determination of d- and l-alanine concentrations using a pyruvic acid sensor. Biosens Bioelectron 18(8):963–971PubMedCrossRefGoogle Scholar
  23. 23.
    Sun H, Yin BH, Ma HL, Yuan HR, Fu B, Liu LB (2015) Synthesis of a novel quinoline skeleton introduced cationic polyfluorene derivative for multimodal antimicrobial application. ACS Appl Mater Interfaces 7(45):25390–25395PubMedCrossRefGoogle Scholar
  24. 24.
    Li LW, Cai ZX, Wu QH, Zhang N, Chen LX, Yu LP (2016) Rational Design of Porous Conjugated Polymers and Roles of residual palladium for photocatalytic hydrogen production. J Am Chem Soc 138(24):7681–7686PubMedCrossRefGoogle Scholar
  25. 25.
    Chen Z, Yuan HX, Liang HY (2017) Synthesis of multifunctional cationic poly(p-phenylenevinylene) for selectively killing bacteria and lysosome-specific imaging. ACS Appl Mater Interfaces 9(11):9260–9264PubMedCrossRefGoogle Scholar
  26. 26.
    Wu SS, Chen CH, Yang HT, Wei W, Zhang YJ, Liu SQ (2018) A sensitive fluorescence “turn-off-on” biosensor for poly (ADP-ribose) polymerase-1 detection based on cationic conjugated polymer-MnO2 nanosheets. Sensors Actuators B Chem 273:1047–1053CrossRefGoogle Scholar
  27. 27.
    Lin YX, Zhou Q, Tang DP, Niessner R, Knopp D (2017) Signal-on photoelectrochemical immunoassay for aflatoxin b-1 based on enzymatic product-etching MnO2 neanosheets for dissociation of carbon dots. Anal Chem 89:5637–5645PubMedCrossRefGoogle Scholar
  28. 28.
    Liu YF, Gao LY, Yan HJ, Shangguan JF, Zhang Z, Xiang X (2018) A cationic conjugated polymer coupled with exonuclease i: application to the fluorometric determination of protein and cell imaging. Microchim Acta 185(2):118CrossRefGoogle Scholar
  29. 29.
    Lu XZ, Jia HX, Yan XH, Wang JS, Wang YC, Liu CH (2017) Label-free detection of histone based on cationic conjugated polymer-mediated fluorescence resonance energy transfer. Talanta 180:150–155PubMedCrossRefGoogle Scholar
  30. 30.
    Wang YH, Jiang K, Zhu JL, Zhang L, Lin HW (2015) A FRET-based carbon dot–MnO2 nanosheet architecture for glutathione sensing in human whole blood samples. Chem Commun 51(64):12748–12751CrossRefGoogle Scholar
  31. 31.
    Mohammadi S, Salimi A (2018) Fluorometric determination of microrna-155 in cancer cells based on carbon dots and MnO2 nanosheets as a donor-acceptor pair. Microchim Acta 185(8):372CrossRefGoogle Scholar
  32. 32.
    Zhao ZL, Fan HH, Zhou GF, Wang RW, Zhang XB, Tan WH (2014) Activatable fluorescence/MRI bimodal platform for tumor cell imaging via MnO2 nanosheet-aptamer nanoprobe. J Am Chem Soc 136(32):11220–11223PubMedPubMedCentralCrossRefGoogle Scholar
  33. 33.
    Yan X, Song Y, Zhu CZ, Song JH, Du D, Lin YH (2016) Graphene quantum dot-MnO2 nanosheet-based optical sensing platform: a sensitive fluorescence “turn off-on” nanosensor for glutathione detection and intracellular imaging. ACS Appl Mater Interfaces 8(34):21990–21996PubMedCrossRefGoogle Scholar
  34. 34.
    Fan HH, Zhao ZL, Yan GB, Zhang XB, Yang C, Meng HM, Chen Z, Liu H, Tan WH (2015) A smart DNAzyme-MnO2 nanosystem for efficient gene silencing. Angew Chem Int Ed 54(16):4801–4805CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  • Ye Lu
    • 1
  • Yong Liu
    • 2
  • Chenchen Wang
    • 1
  • Shuangshuang Wu
    • 1
  • Kai Zhou
    • 1
  • Wei Wei
    • 1
    Email author
  1. 1.Jiangsu Engineering Laboratory of Smart Carbon-Rich Materials and Device, Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, School of Chemistry and Chemical EngineeringSoutheast UniversityNanjingChina
  2. 2.College of Chemistry and Chemical EngineeringHenan UniversityKaifengPeople’s Republic of China

Personalised recommendations