Skip to main content
SpringerLink
Log in

Sensitive Colorimetric Detection of Prostate Specific Antigen Using a Peroxidase-Mimicking Anti-PSA Antibody Coated Au Nanoparticle

  • Original Article
  • Published:
BioChip Journal Aims and scope Submit manuscript

Abstract

The use of colorimetric bioassays for protein detection is one of the most promising diagnostic approaches, but their relatively poor detection limits have been a critical issue. In this study, we developed an efficient colorimetric bioassay based on antibody-coated peroxidase-mimicking Au nanoparticles (Au NPs) for the detection of prostate-specific antigen (PSA), which is one of the most widely used protein biomarkers for the diagnosis of prostate and breast cancers. Anti-PSA antibody adsorption on the Au NP surface result in 65% of the peroxidasemimicking properties of Au NPs was suppressed to maximize sensitivity of the assay. Anti-PSA antibody adsorption on the Au NP was optimized at 150 µg/mL anti-PSA4 antibody for 1 h incubation, and their surface was blocked with 2% BSA. The experimental conditions of the immunoassay detection were also investigated. A highest assay value was obtained at 10 µg/mL anti-PSA1 capture antibody using 3% BSA for surface blocking and at 2.5 pM anti- PSA4-Ab coated Au NPs for 1 h incubation with PSA antigen. As a result, PSA was detected at a wide range of 0.25 to 2,500 ng/mL with a detection limit of 0.23 ng/mL. The anti-PSA-Ab coated Au NP- based assay was simple, easy to operate, and sensitive. The proposed peroxidase-mimicking anti-PSA Ab-coated Au NP-based assay could be further developed to detect other protein biomarkers.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Masson, J.-F. Surface Plasmon Resonance Clinical Biosensors for Medical Diagnostics. ACS Sens. DOI https://doi.org/10.1021/acssensors.6b00763.

    CAS  PubMed  Google Scholar 

  2. Madhavan, B., Yue, S., Galli, U., Rana, S., Gross, W., Müller, M., Giese, N.A., Kalthoff, H., Becker, T., Büchler, M.W. & Zöller, M. Combined evaluation of a panel of protein and miRNA serumexosome biomarkers for pancreatic cancer diagnosis increases sensitivity and specificity. Int. J. Cancer DOI https://doi.org/10.1002/ijc.29324.

    PubMed  Google Scholar 

  3. Xu, J., Shi, M., Chen, W., Huang, Y., Fang, L., Yao, L., Zhao, S., Chen, Z.-F. & Liang, H. A gold nanoparticle-based four-color proximity immunoassay for one-step, multiplexed detection of protein biomarkers using ribonuclease H signal amplification. Chem. Commun. (Cambridge, U. K.) DOI https://doi.org/10.1039/C7CC09404C.

    CAS  PubMed  Google Scholar 

  4. Wang, Y., Dostalek, J. & Knoll, W. Magnetic nanoparticle-enhanced biosensor based on grating-coupled surface plasmon resonance. Anal. Chem. DOI https://doi.org/10.1021/ac200751s.

    CAS  PubMed  Google Scholar 

  5. Nimse, S.B., Sonawane, M.D., Song, K.-S. & Kim, T. Biomarker detection technologies and future directions. Analyst DOI https://doi.org/10.1039/C5AN01790D.

    CAS  PubMed  Google Scholar 

  6. Geißler, D., Stufler, S., Löhmannsröben, H.-G. & Hildebrandt, N. Six-Color Time-Resolved Förster Resonance Energy Transfer for Ultrasensitive Multiplexed Biosensing. J. Am. Chem. Soc. DOI https://doi.org/10.1021/ja310317n.

    PubMed  Google Scholar 

  7. Huang, Y., Liu, X., Huang, H., Qin, J., Zhang, L., Zhao, S., Chen, Z.-F. & Liang, H. Attomolar Detection of Proteins via Cascade Strand-Displacement Amplification and Polystyrene Nanoparticle Enhancement in Fluorescence Polarization Aptasensors. Anal. Chem. 87, 8107–8114 (2015). DOI https://doi.org/10.1021/ac5041692.

    CAS  PubMed  Google Scholar 

  8. Akhavan-Tafti, H., Binger, D.G., Blackwood, J.J., Chen, Y., Creager, R.S., de Silva, R., Eickholt, R.A., Gaibor, J.E., Handley, R.S., Kapsner, K.P., Lopac, S. K., Mazelis, M. E., McLernon, T. L., Mendoza, J. D., Odegaard, B. H., Reddy, S. G., Salvati, M., Schoenfelner, B. A., Shapir, N., Shelly, K. R., Todtleben, J. C., Wang, G. & Xie, W. A homogeneous chemiluminescent immunoassay method. J. Am. Chem. Soc. DOI https://doi.org/10.1021/ja312039k.

    CAS  PubMed  Google Scholar 

  9. Krishnan, S., Mani, V., Wasalathanthri, D., Kumar, C.V. & Rusling, J.F. Attomolar detection of a cancer biomarker protein in serum by surface plasmon resonance using superparamagnetic particle labels. Angew. Chem. Int. Ed. DOI https://doi.org/10.1002/anie.201005607.

    PubMed  Google Scholar 

  10. Munge, B.S., Coffey, A.L., Doucette, J.M., Somba, B.K., Malhotra, R., Patel, V., Gutkind, J.S. & Rusling, J.F. Nanostructured immunosensor for attomolar detection of cancer biomarker interleukin-8 using massively labeled superparamagnetic particles. Angew. Chem. Int. Ed. DOI https://doi.org/10.1002/anie.201102941.

    CAS  PubMed  Google Scholar 

  11. Fang, Y., Li, Y., Zhang, M., Cui, B., Hu, Q. & Wang, L. A novel electrochemical strategy based on porous 3D graphene-starch architecture and silver deposition for ultrasensitive detection of neuron-specific enolase. Analyst DOI https://doi.org/10.1039/C8AN02230E.

    CAS  PubMed  Google Scholar 

  12. Gao, Z., Hou, L., Xu, M. & Tang, D. Enhanced colorimetric immunoassay accompanying with enzyme cascade amplification strategy for ultrasensitive detection of low-abundance protein. Sci. Rep. DOI https://doi.org/10.1038/srep03966

  13. Pham, X.-H., Hahm, E., Kim, T.H., Kim, H.-M., Lee, S.H., Lee, Y.-S., Jeong, D.H. & Jun, B.-H. Enzyme-catalyzed Ag growth on Au nanoparticle-assembled structure for highly sensitive colorimetric immunoassay. Sci. Rep. DOI https://doi.org/10.1038/s41598-018-24664-w.

  14. de la Rica, R. & Stevens, M.M. Plasmonic ELISA for the ultrasensitive detection of disease biomarkers with the naked eye. Nat. Nanotechnol. DOI https://doi.org/10.1038/nnano.2012.186

    CAS  PubMed  Google Scholar 

  15. Li, J., Fu, H.-E., Wu, L.-J., Zheng, A.-X., Chen, G.-N. & Yang, H.-H. General colorimetric detection of proteins and small molecules based on cyclic enzymatic signal amplification and hairpin aptamer probe. Anal. Chem. DOI https://doi.org/10.1021/ac3006186.

    CAS  PubMed  Google Scholar 

  16. Xiao, L., Zhu, A., Xu, Q., Chen, Y., Xu, J. & Weng, J. Colorimetric biosensor for detection of cancer biomarker by Au nanoparticle-decorated Bi2Se3 nanosheets. ACS Appl. Mater. Interfaces DOI https://doi.org/10.1021/acsami.6b15750.

    CAS  Google Scholar 

  17. Song, Y., Wei, W. & Qu, X. Colorimetric biosensing using smart materials. Adv. Mater. DOI https://doi.org/10.1002/adma.201101853.

    CAS  PubMed  Google Scholar 

  18. Zhang, M., Qing, G., Xiong, C., Cui, R., Pang, D.-W. & Sun, T. Dual-responsive gold nanoparticles for colorimetric recognition and testing of carbohydrates with a dispersion-dominated chromogenic process. Adv. Mater. DOI https://doi.org/10.1002/adma.201203289.

    PubMed  Google Scholar 

  19. Lee, J.-S., Lytton-Jean, A.K.R., Hurst, S.J. & Mirkin, C.A. Silver nanoparticle-oligonucleotide conjugates based on DNA with triple cyclic disulfide moieties. Nano Lett. DOI https://doi.org/10.1021/nl071108g.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Gao, Z., Xu, M., Hou, L., Chen, G. & Tang, D. Magnetic bead-based reverse colorimetric immunoassay strategy for sensing biomolecules. Anal. Chem. DOI https://doi.org/10.1021/ac401433p.

    CAS  PubMed  Google Scholar 

  21. Malashikhina, N., Garai-Ibabe, G. & Pavlov, V. Unconventional application of conventional enzymatic substrate: first fluorogenic immunoassay based on enzymatic formation of quantum dots. Anal. Chem. DOI https://doi.org/10.1021/ac4011342.

    CAS  PubMed  Google Scholar 

  22. Babaian, R.J., Johnston, D.A., Naccarato, W., Ayala, A., Bhadkamkar, V.A. & Fritsche, H.A.H.A. Jr. The incidence of prostate cancer in a screening population with a serum prostate specific antigen between 2.5 and 4.0 ng/ml: relation to biopsy strategy. J. Urol. DOI https://doi.org/10.1016/S0022-5347(05)66519-6.

    CAS  Google Scholar 

  23. Bok, R.A. & Small, E.J. Bloodborne biomolecular markers in prostate cancer development and progression. Nat. Rev. Cancer DOI https://doi.org/10.1038/nrc951.

    CAS  PubMed  Google Scholar 

  24. Mannello, F. & Gazzanelli, G. Prostate-specific antigen (PSA/hK3): a further player in the field of breast cancer diagnostics? Breast Cancer Res. DOI https://doi.org/10.1186/bcr302.

    CAS  PubMed  Google Scholar 

  25. Mashkoor, F.C., Al-Asadi, J.N. & Al-Naama, L.M. Serum level of prostate-specific antigen (PSA) in women with breast cancer. Cancer Epidemiol. DOI https://doi.org/10.1016/j.canep.2013.06.009.

    CAS  PubMed  Google Scholar 

  26. Nam, J.-M., Thaxton, C.S. & Mirkin, C.A. Nanoparticle-Based Bio-Bar Codes for the Ultrasensitive Detection of Proteins. Science DOI https://doi.org/10.1126/science.1088755.

    CAS  PubMed  Google Scholar 

  27. Thaxton, C.S., Elghanian, R., Thomas, A.D., Stoeva, S.I., Lee, J.-S., Smith, N.D., Schaeffer, A.J., Klocker, H., Horninger, W., Bartsch, G. & Mirkin, C.A. Nanoparticle-based bio-barcode assay redefines “undetectable” PSA and biochemical recurrence after radical prostatectomy. Proc. Natl. Acad. Sci. U. S. A. DOI https://doi.org/10.1073/pnas.0904719106.

    CAS  Google Scholar 

  28. Chang, H.J., Kang, H.M., Ko, E., Jun, B.H., Lee, H.Y., Lee, Y.S. & Jeang, D.H. PSA Detection with Femtomolar Sensitivity and a Broad Dynamic Range Using SERS Nanoprobes and an Area-Scanning Method. ACS Sens. DOI https://doi.org/10.1021/acssensors.6b00053.

    CAS  Google Scholar 

  29. Chao, C.-H., Wu, C.-S., Huang, C.-C., Liang, J.-C., Wang, H.-T., Tang, P.-T., Lin, L.-Y. & Ko, F.-H. A rapid and portable sensor based on protein-modified gold nanoparticle probes and lateral flow assay for naked eye detection of mercury ion. Microelectron. Eng. DOI https://doi.org/10.1016/j.mee.2012.03.015.

    CAS  Google Scholar 

  30. de L. M. de Morais, C., Carvalho, J.C., Sant’Anna, C., Eugênio, M., Gasparotto, L.H.S. & Lima, K.M.G. A low-cost microcontrolled photometer with one color recognition sensor for selective detection of Pb2+ using gold nanoparticles. Anal. Methods DOI https://doi.org/10.1039/C5AY01762A.

    CAS  Google Scholar 

  31. Kang, J., Zhang, Y., Li, X., Miao, L. & Wu, A. A rapid colorimetric sensor of clenbuterol based on cysteamine-modified gold nanoparticles. ACS Appl. Mater. Interfaces DOI https://doi.org/10.1021/acsami.5b09079.

    Google Scholar 

  32. Rosi, N.L. & Mirkin, C.A. Nanostructures in biodiagnostics. Chem. Rev. DOI https://doi.org/10.1021/cr030067f.

    CAS  PubMed  Google Scholar 

  33. Elghanian, R., Storhoff, J.J., Mucic, R.C., Letsinger, R.L. & Mirkin, C.A. Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles. Science DOI https://doi.org/10.1126/science.277.5329.1078.

    CAS  PubMed  Google Scholar 

  34. Marradi, M., Chiodo, F., García, I. & Penadés, S. Glyconanoparticles as multifunctional and multimodal carbohydrate systems. Chem. Soc. Rev. DOI https://doi.org/10.1039/C2CS35420A.

    CAS  PubMed  Google Scholar 

  35. Zagorovsky, K. & Chan, W.C.W. A plasmonic DNAzyme strategy for point-of-care genetic detection of infectious pathogens. Angew. Chem. Int. Ed. DOI https://doi.org/10.1002/anie.201208715.

    CAS  PubMed  Google Scholar 

  36. Driskell, J.D., Jones, C.A., Tompkins, S.M. & Tripp, R.A. One-step assay for detecting influenza virus using dynamic light scattering and gold nanoparticles. Analyst DOI https://doi.org/10.1039/C1AN15303J.

    CAS  PubMed  Google Scholar 

  37. Lee, C., Gaston, M.A., Weiss, A.A. & Zhang, P. Colorimetric viral detection based on sialic acid stabilized goldnanoparticles. Biosens. Bioelectron. DOI https://doi.org/10.1016/j.bios.2012.10.067.

    CAS  PubMed  Google Scholar 

  38. Liu, Y., Zhang, L., Wei, W., Zhao, H., Zhou, Z., Zhang, Y. & Liu, S. Colorimetric detection of influenza A virus using antibody-functionalized gold nanoparticles. Analyst DOI https://doi.org/10.1039/C5AN00407A.

    CAS  PubMed  Google Scholar 

  39. Zhang, Y., McKelvie, I.D., Cattrall, R.W. & Kolev, S.D. Colorimetric detection based on localised surface plasmon resonance of gold nanoparticles: Merits, inherent shortcomings and future prospects. Talanta DOI https://doi.org/10.1016/j.talanta.2016.02.015.

    CAS  PubMed  Google Scholar 

  40. Liu, L., Hao, Y., Deng, D. & Xia, N. Nanomaterials-based colorimetric immunoassays. Nanomaterials DOI https://doi.org/10.3390/nano9030316.

    CAS  PubMed Central  Google Scholar 

  41. Ronkainen, N.J. & Okon, S.L. Nanomaterial-based electrochemical immunosensors for clinically significant biomarkers. Materials (Basel) DOI https://doi.org/10.3390/ma7064669.

    PubMed  PubMed Central  Google Scholar 

  42. Lee, G., Eom, K., Park, J., Yang, J., Haam, S., Huh, Y.-M., Ryu, J.K., Kim, N.H., Yook, J.I., Lee, S.W., Yoon, D.S. & Kwon, T. Real-time quantitative monitoring of specific peptide cleavage by a proteinase for cancer diagnosis. Angew. Chem. Int. Ed. DOI https://doi.org/10.1002/anie.201108830.

    CAS  PubMed  Google Scholar 

  43. Shi, L., De Paoli, V., Rosenzweig, N. & Rosenzweig, Z. Synthesis and application of quantum dots FRET-based protease sensors. J. Am. Chem. Soc. DOI https://doi.org/10.1021/ja063509o.

    CAS  PubMed  Google Scholar 

  44. Chen, G., Xie, Y., Zhang, H., Wang, P., Cheung, H.-Y., Yang, M., Sun, H. A general colorimetric method for detecting protease activity based on peptide-induced gold nanoparticle aggregation. RSC Adv. DOI https://doi.org/10.1039/C3RA46493H.

    CAS  Google Scholar 

  45. Lee, S.H. & Jun, B.H. Silver nanoparticles: synthesis and application for nanomedicine. Int. J. Mol. Sci. DOI https://doi.org/10.3390/ijms20040865.

    CAS  PubMed Central  Google Scholar 

  46. Park, S.Y., Lee, S.M., Kim, G.B. & Kim, Y.-P. Gold nanoparticle-based fluorescence quenching via metal coordination for assaying protease activity. Gold Bull. (Berlin, Ger.) DOI https://doi.org/10.1007/s13404-012-0070-9.

    CAS  Google Scholar 

  47. Ingram, A., Byers, L., Faulds, K., Moore, B.D. & Graham, D. SERRS-based enzymatic probes for the detection of protease activity. J. Am. Chem. Soc. DOI https://doi.org/10.1021/ja803655h.

    CAS  PubMed  Google Scholar 

  48. Jun, B.H., Kim, G., Jeong, S., Noh, M.S., Pham, X.H., Kang, H., Cho, M.H., Kim, J.H., Lee, Y.S. & Jeong, D.H. Silica core-based Surface-Enhanced Raman Scattering (SERS) tag: advances in multifunctional SERS nanoprobes for bioimaging and targeting of biomarkers. Bull. Korean Chem. Soc. DOI https://doi.org/10.1002/bkcs.10179.

  49. McVey, C., Logan, N., Thanh, N.T.K., Elliott, C. & Cao, C. Unusual switchable peroxidase-mimicking nanozyme for the determination of proteolytic biomarker. Nano Res. DOI https://doi.org/10.1007/s12274-018-2241-3.

    Google Scholar 

  50. Tseng, C.-W., Chang, H.-Y., Chang, J.-Y. & Huang, C.-C. Detection of mercury ions based on mercury-induced switching of enzyme-like activity of platinum/gold nanoparticles. Nanoscale DOI https://doi.org/10.1039/C2NR31716H.

    CAS  PubMed  Google Scholar 

  51. Hvolbæk, B., Janssens, T.V.W., Clausen, B.S., Falsig, H., Christensen, C.H. & Nørskov, J.K. Catalytic activity of Au nanoparticles. Nano Today DOI https://doi.org/10.1016/S1748-0132(07)70113-5.

    Google Scholar 

  52. Deng, H.-H., Weng, S.-H., Huang, S.-L., Zhang, L.-N., Liu, A.-L., Lin, X.-H. & Chen, W. Colorimetric detection of sulfide based on target-induced shielding against the peroxidase-like activity of gold nanoparticles. Anal. Chim. Acta DOI https://doi.org/10.1016/j.aca.2014.09.023.

    CAS  PubMed  Google Scholar 

  53. Zhao, D., Chen, C., Lu, L., Yang, F. & Yang, X. A label-free colorimetric sensor for sulfate based on the inhibition of peroxidase-like activity of cysteamine-modified gold nanoparticles. Sens. Actuators, B DOI https://doi.org/10.1016/j.snb.2015.04.010.

    CAS  Google Scholar 

  54. Hizir, M.S., Top, M., Balcioglu, M., Rana, M., Robertson, N.M., Shen, F., Sheng, J. & Yigit, M.V. Multiplexed activity of perAuxidase: DNA-capped AuNPs act as adjustable Peroxidase. Anal. Chem. DOI https://doi.org/10.1021/acs.analchem.5b03926.

    PubMed  Google Scholar 

  55. Shah, J., Purohit, R., Singh, R., Karakoti, A.S. & Singh, S. ATP-enhanced peroxidase-like activity of gold nanoparticles. J. Colloid Interface Sci. DOI https://doi.org/10.1016/j.jcis.2015.06.015.

    CAS  PubMed  Google Scholar 

  56. Turkevich, J., Stevenson, P.C. & Hillier, J. A study of the nucleation and growth processes in the synthesis of colloidal gold. Discuss. Faraday Soc. DOI https://doi.org/10.1039/DF9511100055.

    Google Scholar 

  57. Haiss, W., Thanh, N.T.K., Aveyard, J. & Fernig, D.G. Determination of size and concentration of gold nanoparticles from UV-vis spectra. Anal. Chem. DOI https://doi.org/10.1021/ac0702084.

    CAS  PubMed  Google Scholar 

  58. Vignati, G., Chiecchio, A., Osnaghi, B., Giovanelli, L. & Meloncelli, C. Different biological matrices (serum and plasma) utilization in consolidation processes: evaluation of seven Access immunoassays. Clin. Chem. Lab. Med. DOI https://doi.org/10.1515/CCLM.2008.032

  59. Liu, Y., Zhang, Z., Yu, J., Xie, J. & Li, C.M. A concentration-dependent multicolor conversion strategy for ultrasensitive colorimetric immunoassay with the naked eye. Anal. Chim. Acta DOI https://doi.org/10.1016/j.aca.2017.01.034.

    CAS  PubMed  Google Scholar 

  60. Suaifan, G.A.R.Y., Esseghaier, C., Ng, A. & Zourob, M. Ultra-rapid colorimetric assay for protease detection using magnetic nanoparticle-based biosensors. Analyst DOI https://doi.org/10.1039/C3AN36881E.

    CAS  PubMed  Google Scholar 

  61. Fu, G., Sanjay, S.T. & Li, X. Cost-effective and sensitive colorimetric immunosensing using an iron oxide-to-Prussian blue nanoparticle conversion strategy. Analyst DOI https://doi.org/10.1039/C6AN00254D.

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Hahn, J., Kim, E., You, Y. & Choi, Y.J. Colorimetric switchable linker-based bioassay for ultrasensitive detection of prostate-specific antigen as a cancer biomarker. Analyst DOI https://doi.org/10.1039/C9AN00552H.

    CAS  PubMed  Google Scholar 

  63. Lai, W., Tang, D., Zhuang, J., Chen, G. & Yang, H. Magnetic bead-based enzyme-chromogenic substrate system for ultrasensitive colorimetric immunoassay accompanying cascade reaction for enzymatic formation of squaric acid-iron(III) chelate. Anal. Chem. DOI https://doi.org/10.1021/ac500738a.

    CAS  PubMed  Google Scholar 

  64. Cao, C., Li, X., Lee, J. & Sim, S.J. Homogenous growth of gold nanocrystals for quantification of PSA protein biomarker. Biosens. Bioelectron. DOI https://doi.org/10.1016/j.bios.2008.07.046.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was supported by the KU Research Professor Program of Konkuk University and funded by Ministry of Science, ICT and Future Planning (NRF-2016M3A9B6918892), and the Korean Health Technology R&D Project, Ministry of Health & Welfare (HI17C1264).

Author information

Authors and Affiliations

  1. Department of Bioscience and Biotechnology, Konkuk University, Seoul, 143-701, Republic of Korea

    Xuan-Hung Pham, Eunil Hahm, Kim-Hung Huynh, Byung Sung Son, Hyung-Mo Kim & Bong-Hyun Jun

Authors
  1. Xuan-Hung Pham
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Eunil Hahm
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kim-Hung Huynh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Byung Sung Son
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hyung-Mo Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bong-Hyun Jun
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bong-Hyun Jun.

Ethics declarations

Conflict of Interests The authors declare no competing financial interests.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pham, XH., Hahm, E., Huynh, KH. et al. Sensitive Colorimetric Detection of Prostate Specific Antigen Using a Peroxidase-Mimicking Anti-PSA Antibody Coated Au Nanoparticle. BioChip J 14, 158–168 (2020). https://doi.org/10.1007/s13206-019-4204-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13206-019-4204-5

Keywords

Search

Navigation