SpringerLink
Log in

An enzymatic nucleic acid vertical flow assay

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Vertical flow assays have been developed in recent years addressing limitations of the lateral flow assays, including limited multiplexing capability, quantitation, and hook effect. In the present study, the first passive paper-based vertical flow assay is developed for the detection of the nucleic acid target. Horseradish peroxidase was used as an enzymatic tracer with a high potential for signal amplification. In order to achieve the best signal-to-noise ratio, different parameters of paper-based assays were optimized. The sample is heat denatured and hybridized with a specific probe to form a dual-labeled hybridization complex. A small volume of diluted sample, 12 µl, can be analyzed within 6 min on the assay in a sandwich format. Assay specificity was evaluated by testing different unrelated samples, and also, 1.7 nM was obtained as the limit of detection (LOD) using the 0 + 3SD method, which is equivalent to 8.5 fmol of double-stranded DNA in the 12 µl sample volume. The linear range of 3–194 nM with a 0.978 correlation coefficient was obtained according to the calibration curve. The developed assay was evaluated with 45 hepatitis B virus clinical plasma samples, and the result showed 100% consistency of the assay with the real-time PCR benchmark. In the present study, we sought to develop a mere detection system for nucleic acid targets, and to investigate the possibility of using enzyme reporter in a passive vertical flow assay.

Graphical abstract

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Ngom B, Guo Y, Wang X, Bi D. Development and application of lateral flow test strip technology for detection of infectious agents and chemical contaminants: a review. Anal Bioanal Chem. 2010;397(3):1113–35.

    Article  CAS  Google Scholar 

  2. Vidic J, Manzano M, Chang C-M, Jaffrezic-Renault N. Advanced biosensors for detection of pathogens related to livestock and poultry. Vet Res. 2017;48(1):1–22.

    Article  Google Scholar 

  3. Liu L, Luo L, Suryoprabowo S, Peng J, Kuang H, Xu C. Development of an immunochromatographic strip test for rapid detection of ciprofloxacin in milk samples. Sensors. 2014;14(9):16785–98.

    Article  Google Scholar 

  4. Old JB, Schweers BA, Boonlayangoor PW, Reich KA. Developmental validation of RSID™-saliva: a lateral flow immunochromatographic strip test for the forensic detection of saliva. J Forensic Sci. 2009;54(4):866–73.

    Article  CAS  Google Scholar 

  5. Li Z, Yi Y, Luo X, **ong N, Liu Y, Li S, Sun R, Wang Y, Hu B, Chen W. Development and clinical application of a rapid IgM-IgG combined antibody test for SARS-CoV-2 infection diagnosis. J Med Virol. 2020;92(9):1518–24.

    Article  CAS  Google Scholar 

  6. Kumanan V, Nugen SR, Baeumner AJ, Chang Y-F. A biosensor assay for the detection of Mycobacterium avium subsp. paratuberculosis in fecal samples. Journal of veterinary science. 2009;10(1):35.

    Article  Google Scholar 

  7. Yang H, Li D, He R, Guo Q, Wang K, Zhang X, Huang P, Cui D. A novel quantum dots–based point of care test for syphilis. Nanoscale Res Lett. 2010;5(5):875–81.

    Article  CAS  Google Scholar 

  8. Tian L, Sato T, Niwa K, Kawase M, Tanner AC, Takahashi N (2014) Rapid and sensitive PCR-dipstick DNA chromatography for multiplex analysis of the oral microbiota. BioMed research international 2014

  9. Kim H-S, Ko H, Kang M-J, Pyun J-C. Highly sensitive rapid test with chemiluminescent signal bands. BioChip J. 2010;4(2):155–60.

    Article  CAS  Google Scholar 

  10. Dodeigne C, Thunus L, Lejeune R. Chemiluminescence as diagnostic tool. A review Talanta. 2000;51(3):415–39.

    Article  CAS  Google Scholar 

  11. Li J, Macdonald J. Multiplexed lateral flow biosensors: technological advances for radically improving point-of-care diagnoses. Biosens Bioelectron. 2016;83:177–92.

    Article  CAS  Google Scholar 

  12. Ross GM, Filippini D, Nielen MW, Salentijn GI. Unraveling the hook effect: a comprehensive study of high antigen concentration effects in sandwich lateral flow immunoassays. Anal Chem. 2020;92(23):15587–95.

    Article  CAS  Google Scholar 

  13. Jiang N, Ahmed R, Damayantharan M, Ünal B, Butt H, Yetisen AK. Lateral and vertical flow assays for point-of-care diagnostics. Adv Healthcare Mater. 2019;8(14):1900244.

    Article  Google Scholar 

  14. Pauli GEN, De La Escosura-Muñiz A, Parolo C, Bechtold IH, Merkoçi A. Lab-in-a-syringe using gold nanoparticles for rapid immunosensing of protein biomarkers. Lab Chip. 2015;15(2):399–405.

    Article  Google Scholar 

  15. Ross G, Salentijn GI, Nielen MW. A critical comparison between flow-through and lateral flow immunoassay formats for visual and smartphone-based multiplex allergen detection. Biosensors. 2019;9(4):143.

    Article  CAS  Google Scholar 

  16. Chen P, Gates-Hollingsworth M, Pandit S, Park A, Montgomery D, AuCoin D, Gu J, Zenhausern F. Paper-based vertical flow immunoassay (VFI) for detection of bio-threat pathogens. Talanta. 2019;191:81–8.

    Article  CAS  Google Scholar 

  17. Nybond S, Réu P, Rhedin S, Svedberg G, Alfvén T, Gantelius J, Svahn HA. Adenoviral detection by recombinase polymerase amplification and vertical flow paper microarray. Anal Bioanal Chem. 2019;411(4):813–22.

    Article  CAS  Google Scholar 

  18. Rivas L, Reuterswärd P, Rasti R, Herrmann B, Mårtensson A, Alfvén T, Gantelius J, Andersson-Svahn H. A vertical flow paper-microarray assay with isothermal DNA amplification for detection of Neisseria meningitidis. Talanta. 2018;183:192–200.

    Article  CAS  Google Scholar 

  19. Clarke O, Goodall B, Hui H, Vats N, Brosseau C. Development of a SERS-based rapid vertical flow assay for point-of-care diagnostics. Anal Chem. 2017;89(3):1405–10.

    Article  CAS  Google Scholar 

  20. Joung H-A, Ballard ZS, Ma A, Tseng DK, Teshome H, Burakowski S, Garner OB, Di Carlo D, Ozcan A. Paper-based multiplexed vertical flow assay for point-of-care testing. Lab Chip. 2019;19(6):1027–34.

    Article  CAS  Google Scholar 

  21. Zhang C, Zhang Y, Wang S. Development of multianalyte flow-through and lateral-flow assays using gold particles and horseradish peroxidase as tracers for the rapid determination of carbaryl and endosulfan in agricultural products. J Agric Food Chem. 2006;54(7):2502–7.

    Article  CAS  Google Scholar 

  22. Mirasoli M, Buragina A, Dolci LS, Guardigli M, Simoni P, Montoya A, Maiolini E, Girotti S, Roda A. Development of a chemiluminescence-based quantitative lateral flow immunoassay for on-field detection of 2, 4, 6-trinitrotoluene. Anal Chim Acta. 2012;721:167–72.

    Article  CAS  Google Scholar 

  23. WHO (2017) Hepatitis B. https://www.who.int/news-room/fact-sheets/detail/hepatitis-b. Accessed 27 Jul 2021.

  24. Locarnini S, Zoulim F. Molecular genetics of HBV infection. Antivir Ther. 2010;15(Suppl 3):3–14.

    Article  CAS  Google Scholar 

  25. Mizokami M, Orito E, Ohba K-i, Ikeo K, Lau JY, Gojobori T. Constrained evolution with respect to gene overlap of hepatitis B virus. J Mol Evol. 1997;44(1):S83–90.

    Article  Google Scholar 

  26. Sapountzi EA, Tragoulias SS, Kalogianni DP, Ioannou PC, Christopoulos TK. Lateral flow devices for nucleic acid analysis exploiting quantum dots as reporters. Anal Chim Acta. 2015;864:48–54.

    Article  CAS  Google Scholar 

  27. Takada K, Sakaguchi Y, Oka C, Hirasawa M. New rapid polymerase chain reaction-immunochromatographic assay for Porphyromonas gingivalis. J Periodontol. 2005;76(4):508–12.

    Article  CAS  Google Scholar 

  28. Jauset-Rubio M, Svobodová M, Mairal T, McNeil C, Keegan N, Saeed A, Abbas MN, El-Shahawi MS, Bashammakh AS, Alyoubi AO. Ultrasensitive, rapid and inexpensive detection of DNA using paper based lateral flow assay. Sci Rep. 2016;6(1):1–10.

    Article  Google Scholar 

  29. Allgöwer SM, Hartmann CA, Holzhauser T. The development of highly specific and sensitive primers for the detection of potentially allergenic soybean (Glycine max) using loop-mediated isothermal amplification combined with lateral flow dipstick (LAMP-LFD). Foods. 2020;9(4):423.

    Article  Google Scholar 

  30. Sajid M, Kawde A-N, Daud M. Designs, formats and applications of lateral flow assay: a literature review. J Saudi Chem Soc. 2015;19(6):689–705.

    Article  Google Scholar 

  31. Gao H, Han J, Yang S, Wang Z, Wang L, Fu Z. Highly sensitive multianalyte immunochromatographic test strip for rapid chemiluminescent detection of ractopamine and salbutamol. Anal Chim Acta. 2014;839:91–6.

    Article  CAS  Google Scholar 

  32. Wang Y, Fill C, Nugen SR. Development of chemiluminescent lateral flow assay for the detection of nucleic acids. Biosensors. 2012;2(1):32–42.

    Article  CAS  Google Scholar 

  33. Posthuma-Trumpie GA, Korf J, van Amerongen A. Lateral flow (immuno) assay: its strengths, weaknesses, opportunities and threats. A literature survey. Analytical and bioanalytical chemistry. 2009;393(2):569–82.

    Article  CAS  Google Scholar 

  34. Chinnasamy T, Segerink LI, Nystrand M, Gantelius J, AnderssonSvahn H. Point-of-care vertical flow allergen microarray assay: proof of concept. Clin Chem. 2014;60(9):1209–16.

    Article  CAS  Google Scholar 

  35. Huang Y, Xu T, Wang W, Wen Y, Li K, Qian L, Zhang X, Liu G. Lateral flow biosensors based on the use of micro-and nanomaterials: a review on recent developments. Microchim Acta. 2020;187(1):1–25.

    Article  Google Scholar 

  36. Zhang Y, San Lee DY, Farwin A, Ying JY. Sieve-through vertical flow platform for efficient liquid exchange in particle-based assays. Anal Chim Acta. 2019;1051:94–102.

    Article  CAS  Google Scholar 

  37. Moumita M, Shankar K, Abhiman P, Shamasundar B. Development of a sandwich vertical flow immunogold assay for rapid detection of oxytetracycline residue in fish tissues. Food Chem. 2019;270:585–92.

    Article  CAS  Google Scholar 

  38. Roda A, Zangheri M, Calabria D, Mirasoli M, Caliceti C, Quintavalla A, Lombardo M, Trombini C, Simoni P. A simple smartphone-based thermochemiluminescent immunosensor for valproic acid detection using 1, 2-dioxetane analogue-doped nanoparticles as a label. Sens Actuators, B Chem. 2019;279:327–33.

    Article  CAS  Google Scholar 

  39. Eltzov E, Marks RS. Colorimetric stack pad immunoassay for bacterial identification. Biosens Bioelectron. 2017;87:572–8.

    Article  CAS  Google Scholar 

  40. Bhardwaj J, Sharma A, Jang J. Vertical flow-based paper immunosensor for rapid electrochemical and colorimetric detection of influenza virus using a different pore size sample pad. Biosens Bioelectron. 2019;126:36–43.

    Article  CAS  Google Scholar 

  41. Oh YK, Joung H-A, Kim S, Kim M-G. Vertical flow immunoassay (VFA) biosensor for a rapid one-step immunoassay. Lab Chip. 2013;13(5):768–72.

    Article  CAS  Google Scholar 

  42. Ramachandran S, Singhal M, McKenzie KG, Osborn JL, Arjyal A, Dongol S, Baker SG, Basnyat B, Farrar J, Dolecek C. A rapid, multiplexed, high-throughput flow-through membrane immunoassay: a convenient alternative to ELISA. Diagnostics. 2013;3(2):244–60.

    Article  CAS  Google Scholar 

Download references

Funding

This work has been conducted under the financial support of the Iran National Science Foundation (INSF) [grant number: 97009535] and Tarbiat Modares University.

Author information

Authors and Affiliations

  1. Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box 14115-111, Tehran, Iran

    Mehdi Tahmasebi & Mehdi Forouzandeh-Moghadam

  2. Department of Medical Virology, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box 14115-111, Tehran, Iran

    Taravat Bamdad

  3. Department of Micro and Nanotechnology, Technical University of Denmark, Ørsteds Plads, 2800 Kgs, Lyngby, Denmark

    Winnie Edith Svendsen

Authors
  1. Mehdi Tahmasebi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Taravat Bamdad
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Winnie Edith Svendsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mehdi Forouzandeh-Moghadam
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mehdi Forouzandeh-Moghadam.

Ethics declarations

Ethics approval

The collection of the samples was approved by the ethics committee of Tarbiat Modares University [Ethic code: IR.TMU.REC.1396.668].

Conflict of interest

The authors declare no competing interests.

Source of biological material

Forty-five clinical plasma samples including 30 hepatitis B–infected patients and 15 healthy volunteers (previously confirmed by the real-time PCR, Qiagen Diagnostics, Hamburg, Germany) were obtained from different clinics around the country.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tahmasebi, M., Bamdad, T., Svendsen, W.E. et al. An enzymatic nucleic acid vertical flow assay. Anal Bioanal Chem 414, 3605–3615 (2022). https://doi.org/10.1007/s00216-022-03988-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-022-03988-7

Keywords

Search

Navigation