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MNAzyme catalyzed signal amplification-mediated lateral flow biosensor for portable and sensitive detection of mycotoxin in food samples

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Abstract

Here, an enzyme-free lateral flow aptasensor was designed by target-induced strand-displacement effect and followed by the activation of multi-component nucleic acid enzyme (MNAzyme)-mediated cleavage to enable rapid and portable ochratoxin A (OTA) detection. The substrate was prepared as an oligonucleotide strand modified with magnetic beads (MB) and human chorionic gonadotropin (hCG). The interaction of OTA with the aptamer induces the release of blocking DNA, which hybridized with three separated subunits of DNA, forming a sequence-specific MNAzyme catalytic core. This core subsequently initiated an enzyme-free MNAzyme cleavage reaction in the presence of the Mg2+ cofactor, cleaving a special substrate and releasing both the incomplete MNAzyme catalytic core and hCG-DNA probe. The incomplete MNAzyme catalytic core was then recognized by substrates once again, triggering a cascade recycling cleavage and resulting in the generation of a larger number of hCG-DNA probes. After magnetic enrichment, the free hCG-DNA probes flow through the pregnancy test strip (PTS) to the T line, generating a colorimetric readout that unequivocally confirms the presence of the target OTA. This work leverages the efficient enzyme-free cleavage amplification of MNAzyme and the PTS-based portable detection device, presenting a biosensing strategy with significant potential for sensitive and portable OTA detection. This method exhibited remarkable sensitivity and selectivity for OTA detection, boasting a detection limit of 5 nM. The present study successfully demonstrated the practical application of this method on real samples, offering a viable alternative for rapid and portable detection of mycotoxins.

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References

  1. Du H, Li X, Xu S, Cheng G, Xue Q, Xu H. N/S-Co-doped carbon dot–based FRET ratiometric fluorescence aptasensing platform modulated with entropy-driven DNA amplifier for ochratoxin a detection. Anal Bioanal Chem. 2023;415(19):4649–60. https://doi.org/10.1007/s00216-023-04778-5.

    Article  PubMed  CAS  Google Scholar 

  2. Malir F, Ostry V, Pfohl-Leszkowicz A, Malir J, Toman J (2016) Ochratoxin A: 50 years of research. Toxins 8 (7). https://doi.org/10.3390/toxins8070191

  3. He Z, Chen Q, Ding S, Wang G, Takarada T, Maeda M. Suppressed DNA base pair stacking assembly of gold nanoparticles in an alcoholic solvent for enhanced ochratoxin A detection in Baijiu. Analyst. 2023;148(6):1291–9. https://doi.org/10.1039/D3AN00016H.

    Article  PubMed  CAS  Google Scholar 

  4. Zhang H, Wang Y, Lin Y, Chu W, Luo Z, Zhao M, Hu J, Miao X, He F. A catalytic hairpin assembly–based Förster resonance energy transfer sensor for ratiometric detection of ochratoxin A in food samples. Anal Bioanal Chem. 2023;415(5):867–74. https://doi.org/10.1007/s00216-022-04479-5.

    Article  PubMed  CAS  Google Scholar 

  5. Yang Y, Li G, Wu D, Liu J, Li X, Luo P, Hu N, Wang H, Wu Y. Recent advances on toxicity and determination methods of mycotoxins in foodstuffs. Trends Food Sci Technol. 2020;96:233–52. https://doi.org/10.1016/j.tifs.2019.12.021.

    Article  CAS  Google Scholar 

  6. Zhu CX, Liu D, Li YY, Ma S, Wang M, You TY (2021) Hairpin DNA assisted dual-ratiometric electrochemical aptasensor with high reliability and anti-interference ability for simultaneous detection of aflatoxin B1 and ochratoxin A. Biosens. Bioelectron. 174. https://doi.org/10.1016/j.bios.2020.112654

  7. Xie X, He Z, Qu C, Sun Z, Cao H, Liu X. Nanobody/NanoBiT system-mediated bioluminescence immunosensor for one-step homogeneous detection of trace ochratoxin A in food. J Hazard Mater. 2022;437: 129435. https://doi.org/10.1016/j.jhazmat.2022.129435.

    Article  PubMed  CAS  Google Scholar 

  8. Yang Y, Su Z, Wu D, Liu J, Zhang X, Wu Y, Li G (2022) Low background interference SERS aptasensor for highly sensitive multiplex mycotoxin detection based on polystyrene microspheres-mediated controlled release of Raman reporters. Anal Chim Acta 1218. https://doi.org/10.1016/j.aca.2022.340000

  9. Zong C, Jiang F, Wang X, Li P, Xu L, Yang H (2021) Imaging sensor array coupled with dual-signal amplification strategy for ultrasensitive chemiluminescence immunoassay of multiple mycotoxins. Biosens. Bioelectron. 177. https://doi.org/10.1016/j.bios.2021.112998

  10. Yang Y, Wu D, Liu J, Su Z, Li L, Wu Y, Li G (2022) High-efficiency enzyme-free catalyzed hairpin assembly-mediated homogeneous SERS and naked-eyes dual-mode assay for ultrasensitive and portable detection of mycotoxin. Biosens Bioelectron. 214. https://doi.org/10.1016/j.bios.2022.114526

  11. Huang Q, Yang H, Wang W, Zhang Y. Multi-target photothermal immunochromatography for simultaneous detection of three mycotoxins in foods. Anal Chim Acta. 2023;1279: 341784. https://doi.org/10.1016/j.aca.2023.341784.

    Article  PubMed  CAS  Google Scholar 

  12. Schax E, Loenne M, Scheper T, Belkin S, Walter J-G. Aptamer-based depletion of small molecular contaminants: a case study using ochratoxin A. Biotechnol Bioprocess Eng. 2015;20(6):1016–25. https://doi.org/10.1007/s12257-015-0486-1.

    Article  CAS  Google Scholar 

  13. Tang Y, Qi L, Liu Y, Guo L, Zhao R, Yang M, Du Y, Li B (2022) CLIPON: a CRISPR-enabled strategy that turns commercial pregnancy test strips into general point-of-need test devices. Angew Chem Int Ed. 61(12). https://doi.org/10.1002/anie.202115907

  14. Rivas L, de la Escosura-Muniz A, Serrano L, Altet L, Francino O, Sanchez A, Merkoci A. Triple lines gold nanoparticle-based lateral flow assay for enhanced and simultaneous detection of Leishmania DNA and endogenous control. Nano Res. 2015;8(11):3704–14. https://doi.org/10.1007/s12274-015-0870-3.

    Article  CAS  Google Scholar 

  15. Qi L, Yang M, Chang D, Zhao W, Zhang S, Du Y, Li Y. A DNA nanoflower-assisted separation-free nucleic acid detection platform with a commercial pregnancy test strip. Angew Chem Int Ed. 2021;60(47):24823–7. https://doi.org/10.1002/anie.202108827.

    Article  CAS  Google Scholar 

  16. Yang M, Tang Y, Qi L, Zhang S, Liu Y, Lu B, Yu J, Zhu K, Li B, Du Y. SARS-CoV-2 point-of-care (POC) diagnosis based on commercial pregnancy test strips and a palm-size microfluidic device. Anal Chem. 2021;93(35):11956–64. https://doi.org/10.1021/acs.analchem.1c01829.

    Article  PubMed  CAS  Google Scholar 

  17. Wang XK, Wang XD, Shi C, Ma CP, Chen LX (2020) Highly sensitive visual detection of nucleic acid based on a universal strand exchange amplification coupled with lateral flow assay strip. Talanta 216. https://doi.org/10.1016/j.talanta.2020.120978.

  18. Zhong ZT, Song LB, Li CQ, Sun X, Chen W, Liu B, Zhao YD (2021) Lateral flow biosensor for universal detection of various targets based on hybridization chain reaction amplification strategy with pregnancy test strip. Sens Actuators B 337. https://doi.org/10.1016/j.snb.2021.129778.

  19. Zhang Y, Ma CB, Yang MT, Pothukuchy A, Du Y. Point-of-care testing of various analytes by means of a one-step competitive displacement reaction and pregnancy test strips. Sens Actuators B. 2019;288:163–70. https://doi.org/10.1016/j.snb.2019.02.091.

    Article  CAS  Google Scholar 

  20. Zhong ZT, Wang HB, Zhang T, Li CQ, Liu B, Zhao YD (2021) Quantitative analysis of various targets based on aptamer and functionalized Fe3O4@graphene oxide in dairy products using pregnancy test strip and smartphone. Food Chem. 352. https://doi.org/10.1016/j.foodchem.2021.129330.

  21. Du Y, Pothukuchy A, Gollihar JD, Nourani A, Li B, Ellington AD. Coupling sensitive nucleic acid amplification with commercial pregnancy test strips. Angew Chem Int Ed. 2017;56(4):992–6. https://doi.org/10.1002/anie.201609108.

    Article  CAS  Google Scholar 

  22. Xu QF, Zhang Y, Xiang DX, Li CC, Zhang CY. A universal DNAzyme-based bioluminescent sensor for label-free detection of biomolecules. Analy Chim Acta. 2018;1043:81–8. https://doi.org/10.1016/j.aca.2018.08.059.

    Article  CAS  Google Scholar 

  23. Khan S, Burciu B, Filipe CDM, Li Y, Dellinger K, Didar TF. DNAzyme-based biosensors: immobilization strategies, applications, and future prospective. ACS Nano. 2021;15(9):13943–69. https://doi.org/10.1021/acsnano.1c04327.

    Article  PubMed  CAS  Google Scholar 

  24. Xie YM, Niu FN, Yu AM, Lai GS. Proximity binding-triggered assembly of two MNAzymes for catalyzed release of G-quadruplex DNAzymes and an ultrasensitive homogeneous bioassay of platelet-derived growth factor. Anal Chem. 2020;92(1):593–8. https://doi.org/10.1021/acs.analchem.9b05002.

    Article  PubMed  CAS  Google Scholar 

  25. Li XM, Cheng W, Li DD, Wu JL, Ding XJ, Cheng Q, Ding SJ. A novel surface plasmon resonance biosensor for enzyme-free and highly sensitive detection of microRNA based on multi component nucleic acid enzyme (MNAzyme)-mediated catalyzed hairpin assembly. Biosens Bioelectron. 2016;80:98–104. https://doi.org/10.1016/j.bios.2016.01.048.

    Article  PubMed  CAS  Google Scholar 

  26. Mokany E, Bone SM, Young PE, Doan TB, Todd AV. MNAzymes, a versatile new class of nucleic acid enzymes that can function as biosensors and molecular switches. J Am Chem Soc. 2010;132(3):1051–9. https://doi.org/10.1021/ja9076777.

    Article  PubMed  CAS  Google Scholar 

  27. Wang Y, Nguyen K, Spitale RC, Chaput JC (2021) A biologically stable DNAzyme that efficiently silences gene expression in cells. Nat Chem 13(4). https://doi.org/10.1038/s41557-021-00645-x.

  28. Hanpanich O, Oyanagi T, Shimada N, Maruyama A (2019) Cationic copolymer-chaperoned DNAzyme sensor for microRNA detection. Biomaterials. 225. https://doi.org/10.1016/j.biomaterials.2019.119535.

  29. Peng HY, Newbigging AM, Wang ZX, Tao J, Deng WC, Le XC, Zhang HQ. DNAzyme-mediated assays for amplified detection of nucleic acids and proteins. Analy Chem. 2018;90(1):190–207. https://doi.org/10.1021/acs.analchem.7b04926.

    Article  CAS  Google Scholar 

  30. Wu D, Wang Y, Zhang Y, Ma H, Pang X, Hu L, Du B, Wei Q. Facile fabrication of an electrochemical aptasensor based on magnetic electrode by using streptavidin modified magnetic beads for sensitive and specific detection of Hg2+. Biosens Bioelectron. 2016;82:9–13. https://doi.org/10.1016/j.bios.2016.03.061.

    Article  PubMed  CAS  Google Scholar 

  31. Mohamed MAA, Kozlowski HN, Kim J, Zagorovsky K, Kantor M, Feld JJ, Mubareka S, Mazzulli T, Chan WCW. diagnosing antibiotic resistance using nucleic acid enzymes and gold nanoparticles. ACS Nano. 2021;15(6):9379–90. https://doi.org/10.1021/acsnano.0c09902.

    Article  CAS  Google Scholar 

  32. Cairns MJ, King A, Sun L-Q. Optimisation of the 10–23 DNAzyme-substrate pairing interactions enhanced RNA cleavage activity at purine-cytosine target sites. Nucleic Acids Res. 2003;31(11):2883–9. https://doi.org/10.1093/nar/gkg378.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Li T, Su Z, Li Y, Xi L, Li G (2022) An aptamer-assisted biological nanopore biosensor for ultra-sensitive detection of ochratoxin A with a portable single-molecule measuring instrument. Talanta. 248. https://doi.org/10.1016/j.talanta.2022.123619.

  34. Zhong ZT, Song LB, He YF, Zhang B, Chen W, Liu B, Zhao YD (2022) Detection of multiple mycotoxins based on catalytic hairpin assembly coupled with pregnancy test strip. Sensors Actuators B 350. https://doi.org/10.1016/j.snb.2021.130911.

  35. Xu G, Zhao J, Yu H, Wang C, Huang Y, Zhao Q, Zhou X, Li C, Liu M. Structural insights into the mechanism of high-affinity binding of ochratoxin A by a DNA aptamer. J Am Chem Soc. 2022;144(17):7731–40. https://doi.org/10.1021/jacs.2c00478.

    Article  PubMed  CAS  Google Scholar 

  36. Cruz-Aguado JA, Penner G. Determination of ochratoxin A with a DNA aptamer. J Agric Food Chem. 2008;56(22):10456–61. https://doi.org/10.1021/jf801957h.

    Article  PubMed  CAS  Google Scholar 

  37. Xiang Y, Belen Camarada M, Wen Y, Wu H, Chen J, Li M, Liao X. Simple voltammetric analyses of ochratoxin A in food samples using highly-stable and anti-fouling black phosphorene nanosensor. Electrochim Acta. 2018;282:490–8. https://doi.org/10.1016/j.electacta.2018.06.055.

    Article  CAS  Google Scholar 

  38. Hu X, Xia Y, Liu Y, Chen Y, Zeng B. An effective ratiometric electrochemical sensor for highly selective and reproducible detection of ochratoxin A: use of magnetic field improved molecularly imprinted polymer. Sensors Actuators B. 2022;359:131582. https://doi.org/10.1016/j.snb.2022.131582.

    Article  CAS  Google Scholar 

  39. Pacheco JG, Castro M, Machado S, Barroso MF, Nouws HPA, Delerue-Matos C. Molecularly imprinted electrochemical sensor for ochratoxin A detection in food samples. Sensors Actuators B. 2015;215:107–12. https://doi.org/10.1016/j.snb.2015.03.046.

    Article  CAS  Google Scholar 

  40. Li S, Kang Y, Shang M, Cai Y, Yang Z. Highly sensitive and selective detection of Ochratoxin a using modified graphene oxide-aptamer sensors as well as application. Microchem J. 2022;179: 107449. https://doi.org/10.1016/j.microc.2022.107449.

    Article  CAS  Google Scholar 

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Funding

This work was supported by the National Natural Science Foundation of China (32022069 and 22076115).

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Authors and Affiliations

  1. School of Biological and Pharmaceutical Engineering, Lanzhou Jiao Tong University, Lanzhou, 730070, China

    Yan Yang

  2. School of Food and Biological Engineering, Shaanxi University of Science and Technology, Xi’an, 710021, China

    Yan Yang, Yiheng Shi, Xianlong Zhang & Guoliang Li

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Yang, Y., Shi, Y., Zhang, X. et al. MNAzyme catalyzed signal amplification-mediated lateral flow biosensor for portable and sensitive detection of mycotoxin in food samples. Anal Bioanal Chem 416, 1057–1067 (2024). https://doi.org/10.1007/s00216-023-05096-6

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